Bimer or an oligomer of a dimer, trimer, quatromer or pentamer of recombinant fusion proteins

ABSTRACT

The invention relates to oligomers of a dimer, trimer, quatromer or pentamer of recombinant fusion proteins. Said oligomers are characterized in that the recombinant fusion proteins have at least one component A and at least one component B, whereby component A contains a protein or a protein segment with a biological function, in particular with a ligand function for antibodies, for soluble or membranous signal molecules, for receptors or an antibody, or an antibody segment, and component B contains a protein or a protein segment which dimerizes or oligomerizes the dimer, trimer, quatromer or pentamer of the recombinant fusion protein, without the action of third-party molecules. The invention also relates to the use of dimers or oligomers of this type for producing a medicament, to the fusion proteins which cluster in dimers or oligomers and to their DNA sequence and expression vectors or host cells comprising this DNA sequence.

The present invention concerns bimers or oligomers of bimers, trimers,quadromers or pentamers of recombinant fusion proteins, whereby therecombinant fusion proteins have at least one component A and onecomponent B. In addition, the present invention concerns the use ofbimers or oligomers of these types to manufacture a drug and/or theiruse for in-vitro diagnosis. Finally, the present invention also concernsfusion proteins that have one component A and one component B, wherebythe component B contains a multimerizing and oligomerizing segment, or afunctional derivative of a segment of this type of a protein, selectedfrom the group consisting of the family of C1q proteins or thecollections. DNA sequences that encode for a fusion protein of this typeand expressions vectors and host cells which contain the DNA sequenceand/or the expression vector are also the subject of the presentinvention.

Proteins, which occur physiologically as dimers, trimers, quadromers orpentamers, are found in great numbers in nature. Because of theinteractions at the surfaces of these proteins which dimerize ormultimerize in solution, there may be spontaneous aggregation ofproteins or even, as well, for example, aggregation that is kineticallydelayed because it is dependent on the concentration or the milieu. Thecauses of this are hydrophobic interactions, hydrogen bond formationsand/or coulomb forces.

However, together with this, structure motives are found with certainproteins that lead to the formation of specific structuralsupersecondary structures and thus to protein dimers or multimers. Theformation of supersecondary structures is based on characteristic aminoacid sequences of the proteins that form these dimers or multimers.“Coiled-coil helices” might be referred to as supersecondary structures,for example, which effect a dimerizing or multimerizing of proteinsthrough interactions of characteristic α-helices, which occur with eachof the proteins that form the coiled-coil form. The coiled-coil helix asan intermolecular “dimerizing or multimerizing domain” of proteinsexhibits structurally a super helix with two or more helices coiledaround themselves. These types of coiled-coil motives are found inparticular with extracellular protein dimers or multimers, and inparticular with proteins or protein complexes of the connective tissue.

Beck et al. (J. Mol. Biol. (1996) 256, 909-23) for example describe aconnective tissue protein, the so-called cartilage matrix protein (CMP),whose aggregation to a homotrimer on triple helix, which is the resultof the aggregation of three complementary helices (each as components ofa polypeptide), is based on the coiled-coil pattern. Characteristic forthe amino acid sequence of a helix of this type forming a triple helixis the heptad pattern (abcdefg)_(n). The latter's amino acids in thepositions a and d usually carry nonpolar side chains and thus permit theformation of the superhelical structure described above, here as atriple helix made of three helices.

In addition, the literature also states that with another extracellularmatrix protein (cartilage oligomeric matrix protein) (COMP) there are infact five helices in the form of a five-coil coiled-coil helix thatinteract with one another and thus are able to form pentamers. (Kajava,PROTEINS: Structure, Function and Genetics, 24:218-226 (1996);Malashkevich et al., Science, Vol. 274, 761-765, 1996).

Along with the matrix proteins COMP and CMP, which do not belong to theproteins from the collagen family, specific structural multimerizingphenomena through the formation of supersecondary structures are alsofound with proteins from the collagen family. Here the structure ofcollagen fibres is characterized by the tropocollagen that consists ofthree helically twisted polypeptides. The protofibrilla of a hair isalso developed from a triple helix of α-keratin with the motive“coiled-coil”, although in this case left-handed.

To increase the avidity of ligands, Terskikh et al. (PNAS, Vol. 94,1663-1668, 1997) suggested using fusion proteins with a short peptidewith ligand function and the “coiled-coil” domain of the matrix proteinCOMP. An increased avidity could be verified for these pentamers,whereby aggregates of a higher order cannot be obtained in this way.

In addition, because of their sequence homologies in their respectivemultimerizing sequence sections, the proteins C1q, collagens α1 (X), α2(VII), the hibernating protein, ACRP30, the internal ear structureprotein, cerebellin and multimerin as protein family are broughttogether under the designation C1q family (Kischore and Reid,Immunopharmacol., 42 (1999) 15-21), which is found structurally asdimers or multimers. Among the proteins with multimerizingcharacteristics occurring in this family, for example, the structure ofthe protein C1q, which is familiar from the complement system, ischaracterized by monomers which each have a globular, so-called headdomain, and a “collagen-like” helical sequence section. The monomerstrimerize through this helical sequence section, which forms acoiled-coil triple helix. Six of these C1q trimers themselves form anoligomer, whereby the oligomerizing of the protein trimers is based oninteractions between the individual coiled-coil triple helices. Theresult is that this structural arrangement in the protein or themultimerized (oligomerized) protein complex C1q leads to a structurereferred to as a “bouquet”, whereby it is ensured that 18 globular,C-terminally arranged head domains are linked to a hexamer of trimers.

A similar structure as with protein C1q can be also seen in the proteinACRP30, which is also a protein from the C1q family (Hu et al., J. Biol.Chem., Vol. 271, No. 18, 10697-10703, 1996). This serum protein, whichis secreted by adipocytes, is in all probability quadromers of trimers,whereby, as with the C1q protein, globular C-terminal domains are linkedvia triple helices similar to collagens. Probably four of these triplehelices finally form an oligomer themselves through correspondinginteractions. In the publication by Shapiro and Scherer (Current Biology1998, 8:335-338) the structure of a homotrimer of ACRP30 is shown thatwas determined with the help of X-ray structural analysis.

In addition, proteins from the class of the collectins are known fromthe literature which are characterized by a collagen-like domain, a neckregion and in addition by a globular carboxyterminal lectin-bindingdomain. The collectins also occur physiologically as oligomers oftrimers. For example, the lung surfactant protein A (SP-A) and themannose binding protein (MBP), both of which are from the family ofcollectins, trimerize through the interactions of their “collagen-like”domains and are finally found as hexamers of trimers (Epstein et al.,Current Opinion in Immunology, Vol. 8, No. 1, 1996, 29-35). Accordingly,the proteins known under the designation of collectins form oligomers(e.g. hexamers) of multimers (e.g. trimers).

The literature also shows that numerous proteins that have aphysiological effect as signal molecules can only transduct a biologicalsignal under certain conditions. For example, membrane bound FasL isbiologically, i.e. apoptotically, effective, whereas after the cleavingof the extracellular protein segment (so-called FasL) thisnon-membrane-bound sFasL fraction can no longer bring about an apoptoticeffect on target cells. The publication by Schneider et al. (J. Exp.Med., Vol. 187, No. 8, 1998, 1205-1213) states that the biologicaleffect of sFasL trimers which, as explained previously, are obtainedafter cleaving from membrane-bound protein segment, can in fact bereactivated with regard to their physiological function through the useof crosslinking antibodies. For this purpose a fusion protein wasconstructed that consists of the trimerizing domain of FasL, a shortlinker sequence and a flag marking (with the flag amino acid sequence(single-letter code) DYKDDDDK), expressed, and this type of fusionprotein which is non-structurally trimerized (i.e. not through specificsecondary structure interactions with the result of the formation of asupersecondary structure) was crosslinked through antibodies directedagainst the flag tag.

This type of sFasL molecules crosslinked through antibody bindingdisplays a significant increase of the specific apoptotic activity asagainst non-crosslinked sFasL trimers. This procedure, which issuggested by Schneider et al., does however have the disadvantage that,along with the recombinant, non membrane-bound FasL proteins with thetrimerizing domain, specific antibodies also have to be used, in otherwords, an increase in biological activity can only be achieved throughthe provision of an additional molecule fraction. In addition, with thetheory suggested by Schneider et al. it is not possible to ensure anexactly preset or determinable degree of oligomerizing of the multimers.The antibodies can namely have the effect that the FasL trimersassociate to dimers or even that a wide spectrum of oligomerizedcomplexes through to huge sFasL/antibody aggregates occurs. Because anexactly defined product with maximum efficacy is required, for examplefor medical applications, the result is that the way proposed bySchneider et al. for reactivating and/or increasing sFasL activity, isnot practical.

A central object of the present invention is therefore to providecompounds which avoid the disadvantages of the state of the art, inparticular which display increased biological activity or bring about areactivation of the biological activity.

The present object is solved by the subject-matter of Claim 1, namelybimers or oligomers of a dimer, trimer, quadromer, or pentamer ofrecombinant fusion proteins, in that the recombinant fusion proteinshave at least one component A and at least one component B, wherebycomponent A covers a protein or a protein segment with biologicalfunction, in particular with a binding function, and component B coversa protein or a protein segment which bimerizes or oligomerizes thedimer, trimer, quadromer, or pentamer of a recombinant fusion proteinwith biological function without the effect of tertiary molecules, oraggregates fusion proteins to dimers or multimers and at the same timelinks these dimers or multimers together to a bimer or oligomer withoutthe effect of tertiary molecules.

In the representation of the present invention the terms dimer, trimer,quadromer, or pentamer are summarized under the designation multimer andthis will be understood as protein complexes from two, three, four orfive associated polypeptides (proteins). In contrast, the aggregates ofthe next higher order, that is, the aggregations of two or more dimers,trimers, quadromers, or pentamers in the above sense are referred to asbimers or oligomers. Proteins or protein segments with biologicalfunctions (component A in the fusion protein) are understood inparticular to be proteins which have a ligand function, particularly foranti-bodies or receptors (i.e. can occur in interaction as a bindingpartner with one or more molecules), modified amino acid sequences, e.g.amino acid sequences with covalent or non-covalent coupled effectiveagents (possibly of a organic-chemical nature), antibodies or segmentsof antibodies with paratopes or even hormones, for example, peptidehormones. In particular, the present invention encompasses amino acidsequences of signal proteins as component A in the fusion protein whichare biologically already active as monomers and whose effect isincreased accordingly as components in a complex according to thepresent invention, or which only become active through the multimerizingor oligomerizing initiated in accordance with the present invention orthrough the oligomerizing initiated exclusively in accordance with thepresent invention (in so far as component A of the fusion protein isalready found as a trimer). With physiologically membrane-bound signalproteins, e.g. with TNF cytokines, cleavage products are preferred whichcontain the extra-membranous, in particular the extra-cellular, proteinsegments. But amino acid sequences which can function as antigens canalso be used as component A in a recombinant fusion protein. Finally,receptors, e.g. receptors from the TNF receptor family, e.g. belongingto the family of type I membrane proteins (e.g. FasR), or segments orderivatives of such receptors, can also be used as component A, whichalso have a binding function (i.e. interact as a binding partner withanother molecule) and therefore fall under the term “ligand” within themeaning of the present invention. These types of capable of bindingfragments of biological receptors are suitable in particular for use asdrugs, if the complementary biological ligand is found in the patient innon-physiologically high concentrations.

In a preferred embodiment the components A can have the multimers foundin the oligomers in accordance with the present invention, i.e., dimers,trimers, quadromers, or pentamers, identical components A (oligomers ofhomodimers or homomultimers) or different components A (oligomers ofheterodimers or heteromultimers). In this way, proteins with differentcomponents A, possibly also with a different biological function, can belinked together in dimers or multimers of oligomers in accordance withthe present invention. The individual heterodimers or heteromultimersaggregated in the bimers or oligomers can also be the same or different,i.e. a bimer or oligomer in accordance with the present invention mayalso be composed of different heterodimers or heterooligomers.

However, it is also possible that the fusion proteins in the respectivedimer or multimer as a subunit of the bimer or oligomer are identical,but, on the other hand, the individual subunits in the bimer or oligomerarranged as a dimer or multimer are different (heterobimer orheterooligomer of homodimers, homotrimers, homoquadromers orhomopentamers). In this way, for example, up to six homotrimers that aredifferent with regard to component A can be associated in a hexamer oftrimers in accordance with the present invention. In this way, typicallyprecisely modulated biological activities can be brought about by theselection, the arrangement, the specific combination and/or through thenumber of components A in the bimer or oligomer. It is known thatcertain biological effects bring about the desired biological effect,e.g. a cell activation, only through the interaction of at least twoligands (in the biological sense, not in the extended meaning inaccordance with the present invention). This is desirable, e.g. with thecombination of certain interleukines with regard to the effect as T-cellor B-cell activators. In accordance with the present invention,effectors of this type which are only effective in combination can bearranged in a complex in accordance with the present invention. However,it is also conceivable that compositions will be provided that, forexample with regard to the respective component A, contain differentoligomers.

In another preferred embodiment the component A in a recombinant fusionprotein is a peptide hormone, a growth factor, a cytokine, aninterleukin or a segment of these, preferably a segment capable ofbinding. However, functional derivatives of the above-mentioned peptidesand/or proteins can also be used as component A in the recombinantfusion protein which is a component of an oligomer in accordance withthe present invention.

Proteins in particular which maintain the biological function, but atthe same time have sequence differences to the corresponding nativesequences, are described as functional derivatives of biologicallyactive proteins protein segments or peptides. The sequential deviationsmay be one or more insertions, deletions or substitutions, whereby asequence homology of at least 70% is preferred and a sequence homologyof at least 85% between the derivative used and the native sequence isparticularly preferred. Those amino acid sequences in particular comeunder the term “functional derivatives” which display conservativesubstitutions as against the physiological sequences. Conservativesubstitutions are taken to be those substitutions in which amino acidswhich come from the same class are substituted for one another. Thereare in particular amino acids with aliphatic side chains, positively ornegatively charged side chains, aromatic groups in the side chains, oramino acids whose side chains can be part of hydrogen bonds, forexample, side chains with a hydroxy function. This means that, forexample, an amino acid with a polar side chain is replaced by anotheramino acid also with a polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid which also has a hydrophobic side chain (e.g. serine(threonine) by threonine (serine), or leucine (isoleucine) by isoleucine(leucine).

In accordance with the present invention a ligand is understood to beall molecules that take part in binding reactions. A ligand cantherefore be a protein that is normally described as a receptor. Areceptor of this type can also be a “ligand” within the meaning of thepresent invention if it binds a signal molecule.

Under the present invention, oligomers of trimers of recombinant fusionproteins are preferred, in particular trimers or quadromers of trimers(3×3 or 4×3) or hexamers of trimers (6×3).

Particularly preferred is a bimer or oligomer of a dimer, trimer,quadromer, or pentamer of recombinant fusion proteins when component Ain the recombinant fusion protein is a cytokine from the TNF cytokinefamily, a segment of this type of TNF cytokine or a functionalderivative of a TNF cytokine or of a corresponding TNF cytokine segment.Here the TNF cytokines that are used can lead to, for example,apoptotic, proliferating or activating effects in the target cells bybinding to the corresponding receptors. In a non-exhaustive list, theproteins CD40L, FasL, TRAIL, TNF, CD30L, OX40L, RANKL, TWEAK, Lta,Ltab2, LIGHT, CD27L, 41-BB, GITRL, APRIL, EDA, VEGI and BAFF can inparticular be considered for use as TNF cytokines. Extracellularsegments of the above-mentioned membrane-bound TNF-cytokines or otherfunctional derivatives are preferred for use as component A inrecombinant fusion proteins. These cleavage products are particularlypreferred when their respective biological functionality, in particulartheir capacity for binding to the respective receptor, is retained.Functional derivatives in the above sense of the above-mentioned TNFcytokines or segments of TNF cytokines can also be used as component Aof the fusion protein. In a particularly preferred embodiment, componentA of the recombinant fusion protein is chosen from the group consistingof hFasL (AA 139-261), hTRAIL (AA 95-281), hCD40L (AA 116-261) and m orhTNFα (AA 77-235).

In addition, receptors (membrane-bound or extracellular), in particularreceptors of proteins of the family of the TNF cytokines, in particularthe physiological receptors of the above-mentioned TNF cytokines orsegments or derivatives of the receptors are used in a preferredembodiment as component A in the recombinant fusion protein. In theevent that segments of receptors are used as component A these will inparticular be segments of the physiological protein sequence of thesetypes of receptors which are arranged physiologicallyextra-membranously. The extracellular segments of these type ofreceptors come in particular into consideration here. For example, inaccordance with the present invention the binding domain(s) of areceptor, in particular of a receptor which binds a cytokine from thefamily of the TNF cytokines (e.g. FasR, CD30, CD40, GITR, TNF-R1 and/orTNF-R2), can be provided on a dimerizing immunoglobulin (dimerizing Fcfragment) and these dimers can themselves be bimerized or oligomerizedto bimer or oligomer complexes in accordance with the present inventionthrough a component B, for example a collagen-like segment with thecapability of bimerizing or oligomerizing dimers or multimers. For thispurpose, for example, a tetramer of dimers or multimers (e.g. throughtetramerizing segments of ACPR30) may be considered, or a pentamer ofdimers or multimers (e.g. through corresponding sequence sections of amonomer from the COMP complex used as component B) or even a hexamer ofdimers or multimers (e.g. through hexamerizing segments from monomers ofthe C1q complex).

Under the present invention the following possibilities are given: thecomponent A which is selected for a recombinant fusion protein, which isto become a component of an oligomer in accordance with, the presentinvention, is already found as such in solution as a dimer or multimer.The component B in such a case will only intensify the dimerizing ormultimerizing of component A and will essentially lead to the bimerizingor oligomerizing of the recombinant fusion proteins. This situation isfound, for example, if, as component A, at least one TNF ligand or asegment or derivative of the same, which is already typically trimerizedin solution, is to be oligomerized as component(s) of a fusion protein.However, in the event that component A as such in solution does not showany dimerizing or multimerizing mediated by surface interaction, inaccordance with the present invention component B must ensure not onlydimerizing or multimerizing of component A but also bimerizing oroligomerizing of the dimerized or multimerized recombinant fusionproteins. This is typically necessary, for example, for the case thatreceptors or segments thereof form the component A in the recombinantfusion protein.

In the framework of the present invention bimers or oligomers of dimers,trimers, quadromers, or pentamers of recombinant fusion proteins aredisclosed in which component A is preferably an antigen or a segment ofan antigen. It is desirable here to use antigens from viral, bacterialor, protozoological pathogens. These may be any typical antigen of apathogen, for example, protein segments and/or specific carbohydratestructures, but they are typically surface antigens of the respectivepathogens or segments of surface proteins of pathogens which alsodisplay antigenic properties. For example, the following non-exhaustiveexamples might conceivably be used: haemagglutinin, neuraminidase,PreS1, PreS2, HBs antigen, gp120, gp41 or even typical tumour antigens.

In a preferred embodiment component A of the recombinant fusion proteinmay also be an amino acid sequence which is suitable for acting as acarrier for a receptor agonist or receptor antagonist. For example, asmall organic-chemical molecule active as a pharmacological agent can betypically coupled covalently to this type of amino acid sequence, forexample through an ether bond to threonine or serine, an amid-like bondor through an ester bond. Through the present invention large oligomercomplexes of for example 18 fusion proteins (e.g. 3×6 fusion proteins)are made available each with connected receptor agonists or receptorantagonists. In this way, it is possible, to achieve a considerableimprovement of the efficacy or of the avidity of these types oforganic-chemical molecule at their respective receptors, placed on abimeric or oligomeric protein carrier, for example for use as a drug inhuman or veterinary medicine.

Component B of the recombinant fusion proteins, which is found dimerizedor, multimerized in the bimer or oligomer, is typically a protein fromthe family of the C1q proteins or the collectins. Particularly preferredare the proteins of the C1q proteins or the collectin family as acomponent of the recombinant fusion proteins, namely as component B ifonly their dimerizing/multimerizing sequence or bimerizing/oligomerizingsequence in the recombinant fusion protein is transcribed or translated.The mainly globular head domains (FIG. 14), which are contained in thesequence of native monomers, will therefore, as a translation product,not appear in the recombinant fusion protein in accordance with thepresent invention and are therefore not a component of component B inthis protein. The above-mentioned component B in a recombinant fusionprotein in accordance with the present invention will show a sequencewhich typically mainly overlapping has the functionality fordimerizing/multimerizing or bimerizing/oligomerizing respectively,because the collagen-like segments of the proteins of theabove-mentioned families used as component B participate typically inthe formation of, for example, triple helices, which themselves have thecapability to enter into a bimer or oligomer structure (for example, atetramer or hexamer of, for example, triple helices) with other triplehelices.

Typically therefore the multimerizing and oligomerizing fusion proteinwill have as component B the domains of the proteins from the familiesof the C1q proteins or collectins which are responsible for thedimerizing and multimerizing and/or the bimerizing and oligomerizing,while their respective head domains are replaced as component A by otherproteins or protein segments which also carry out a biological function.The term “recombinant fusion protein” is therefore to be understood inthe framework of the present invention as the minimum one component Aand the minimum one component B in the recombinant fusion protein beingartificially fused, i.e., that a fusion protein within the meaning ofthe present invention does not correspond to a naturally occurringprotein.

Functional, i.e. bimerizing or oligomerizing derivatives of proteinsfrom the C1q family or the family of collectins, or derivatives ofsegments of the above-mentioned proteins can also be used as component Bfor the aggregation of recombinant fusion proteins to bimers oroligomers. In this case, for example, the component B will contain thesequence of the protein C1q, MBP, SP-A (lung surfactant protein A), SP-D(lung surfactant protein D), BC (bovine serum conglutinin), CL43 (bovinecollectin-43) and/or ACRP30, or the sequence(s) of bimerizing oroligomerizing segments of at least one of the above-mentioned proteinsor of functional derivatives of these proteins of or the segments of thefunctional derivatives. Bimers or oligomers of recombinant fusionproteins are particularly preferred when component B of the recombinantfusion protein is a protein segment of the protein C1q or the proteinACRP30, in particular of a human variant or mammalian variant, moreparticularly of the murine variant, whereby a respective protein segmentof this type typically does not have a globular head domain of thenative protein C1q or protein ACRP30.

An extremely preferred embodiment of the present invention isrepresented by bimers or oligomers of dimers, trimers, quadromers, orpentamers of recombinant fusion proteins whose component B contains anamino acid sequence in accordance with FIG. 6A (framed sequence) or FIG.6B or a functional derivative of this/these amino acid sequence(s)and/or a segment of this/these sequence(s). Typically, this sequence isa segment of the protein mACRP30 (m: murine), e.g. with the amino acids18 to 111, or a segment of the human variant (hACRP30), e.g. with aminoacids 18 to 108. In particular, according to the present invention afusion protein can therefore be provided whose components A and B are ofhuman origin such that possible immune reactions in humans can beexcluded during therapeutical application. Particularly preferred arebimers of oligomers of dimers or multimers of those fusion proteins thathave sequences from different host organisms. Aggregates in accordancewith the present invention are extremely preferred if they stem fromchimary fusion proteins, whereby component A stems from a different typeof animal to component B. It can be advantageous if component Acorresponds to an amino acid sequence from a mouse, rat, pig or othervertebrate, in particular from a mammal, or to a functional derivativeof the same, and component B is of human origin, or vice versa. E.g.complexes in accordance with the present invention of those proteinswhose component A corresponds to a sequence from a virus, bacterium orprotozoon, combined with a component B of human origin, are alsopreferred. Naturally, the sequences of component A and component B in afusion protein in accordance with the present invention can also stemfrom the same type of animal.

In a further preferred embodiment of the present invention themultimerizing and/or oligomerizing of the fusion protein takes placethrough a short amino acid sequence of more than 6 amino acids,preferably between 8 and 20 amino acids, which is present in therecombinant fusion proteins as component B. The bimerizing oroligomerizing of fusion proteins, which are already found as such notthrough supersecondary structures but through surface interaction insolution as dimers or multimers, which is typically achieved throughthis short amino acid sequence, is preferably based on the formation ofdisulphide bridges, which is possible through the specific amino acidsequence in the recombinant fusion protein. This means that component Bpreferably has at least one cystein, which under oxidizing conditionscan form a covalent link with the at least one cystein of a fusionprotein of at least one other dimer or multimer. The amino acid sequence(single-letter code) VDLEGSTSNGRQCAGIRL would be an example of thepreferred case that component B contains a short bimerizing oroligomerizing amino acid sequence of between 8 and 20 amino acids. Thissequence of 18 amino acids has a cystein residue at position 11 whichcan form a disulphide bridge between the dimers or multimers.

Functional derivatives or segments of these 18 amino acids containingsequences can be used as component B. Here the sequenceVDLEGSTSNGRQSAGIRL should be mentioned in particular, which, althoughthe cystein residue at position 11 has been substituted by serineresidue, can still ensure bimerizing or oligomerizing of the fusionprotein multimers.

The fusion proteins can be arranged in a preferred embodiment in such away that aggregates of a higher order can be formed beyond thebimerizing or oligomerizing of dimerized or multimerized fusionproteins, This higher order aggregates, which themselves comprise two ormore bimers or oligomers, can be provided, for example, throughantibodies via crosslinking. The antibodies are directed againstepitopes on the fusion protein(s) of a complex in accordance with thepresent invention, preferably against an epitope of component B.However, together with component A and component B the fusion proteincan also have additional sequence sections which serve as antigens forthe crosslinking antibodies. In this context, so-called tag sequencesare preferred in the framework of the present invention, for example aflag tag, in other words the amino acid sequence DYKDDDDK, or also, forexample, a His tag (containing several consecutive histidines).

However, special preference in accordance with the present invention isgiven to the provision of aggregates of a higher order through more thanone component B being contained in the recombinant fusion protein.Preferably, for the formation of aggregates of a higher order, thefusion protein will contain a component B1 for the formation of bimersof oligomers and in addition at least one other component B (preferablya component B2) which is typically different from component B1. Thecomponent B2, which must be found in at least one fusion protein of abimer or oligomer in accordance with the present invention, ensures thatthe bimers or oligomers form aggregates of a higher order. For example,component B1 can be a bimerizing or oligomerizing segment of a proteinfrom the family of the C1q or collectin proteins, while component B2 isa sequence with 8 to, for example, 28 amino acids which forms at leastone disulphide bridge. In the event that at least one disulphide bridgeis formed between two different oligomers, a higher order aggregate inaccordance with the present invention will be made available, forexample a bimer of an oligomer.

In addition, preference will be given to the insertion of so-calledlinker sequences between component A and component(s) B or, if there areseveral components B in the fusion protein, between these minimum twocomponents. These linker sequences allow structural separation of thedifferent functional components in the recombinant fusion protein andcan preferably take over a “hinge” function, i.e. represent an aminoacid sequence with a flexible structure.

This discloses in general in accordance with the present inventionbimers or oligomers or aggregates of a higher order which arecharacterized by an increased biological efficiency and/or throughincreased avidities at complementary proteins. In this way, as a furthersubject-matter of the present invention, methods are disclosed whichserve the increase of biological efficiency and/or the increase of theavidity of biomolecules or drugs with ligand functions in the sense ofthe present invention. Methods of this type are characterized by therecombination of at least one component A, which corresponds to aprotein or protein segment with biological function, with at least onedimerizing or multimerizing and bimerizing or oligomerizing component B,whereby an increase in the biological activity and/or an increase in theavidity of component A is achieved by the recombinant fusion proteinsfinally being bimerized or oligomerized through multimerizing andoligomerizing to bimers or oligomers of dimers and multimers. The methodis highly preferred when component A is a TNF cytokine, a segment of aTNF cytokine or a functional derivative of such a protein or proteinsegment. A further preferred method is the recombination of at least onecomponent A with at least one component B into a recombinant fusionprotein, whereby at least one component A is a receptor or a segment ofa receptor, preferably of a TNF receptor. With regard to preferredembodiments of a method of this kind in accordance with the presentinvention, the preferred embodiments for the bimers or oligomersdescribed above apply analogously.

The bimers or oligomers of the present invention may be used for theproduction of a drug or for the treatment of illnesses or disorders inmedical use, i.e. for both human and veterinary medicine. The higherorder aggregates formed in accordance with the present invention frombimers or oligomers are also suitable for use as drugs or for theproduction of a drug. A wide range of illnesses or disorders can betreated with bimers or oligomers or with the higher order aggregatesclaimed in accordance with the present invention. This type of bimers oroligomers or higher order aggregates can be used to produce a drug forthe treatment of the following list of illnesses or disorders which isby no means exhaustive: hyperinflammatory disorders, autoimmunediseases, illnesses based on hyperapoptotic or hypoapoptotic reactions,neurodegenerative diseases, but also for treating infectious diseases,tumours, and/or endocrinological disorders. With regard to infectiousdiseases the use of bimers and oligomers with bacterial orprotozoological diseases, but in particular with viral infections, is tobe referred to, whereby antibodies or segments of antibodies carryingparatopes are particularly preferred here as component A in therecombinant fusion protein. Bimers or oligomers or their higher orderaggregates are specially suitable where the disease makes treatment withbiologically active cytokines necessary, for example including for thetreatment and/or the production of a drug for the treatment of tumours,in particular of tumours of the lymphatic system.

In addition, the bimers or oligomers in accordance with the presentinvention will also be used as vaccines or for the production of avaccine for active or passive immunization against infectious diseases.In the case of active immunization an antigen suitable for vaccinationwill be used as component A in the recombinant fusion protein. Inparticular, surface antigens or segments of surface antigens ofbacteria, viruses or protozoa, e.g. of plasmodia or trypanosomes, can beused. In a non-exhaustive list, a vaccine which contains bimers oroligomers or aggregates of these, whereby the recombinant fusionproteins must have at least one component A, in other words one or moreidentical or different antigens of the pathogen, can be used tovaccinate against German measles, measles, poliomyelitis, rabies,tetanus, diphtheria, BCG, tuberculosis, malaria, yellow fever, HIV orinfluenza viruses, for example rhinoviruses. The combination ofdifferent antigens in a bimer or oligomer or a higher order aggregateformed from bimers or oligomers is also possible in accordance with thepresent invention, whereby different antigens from the same pathogen canbe combined in a bimer or oligomer, or antigens from two or morepathogens can be combined in a bimer or oligomer or in a higher orderaggregate. Typically, two or more components A1, A2 to AX can becontained in a fusion protein, which is then a component of a bimer oroligomer or of a higher order aggregate in accordance with the presentinvention, or two or more fusion proteins that are different with regardto at least one component A can be combined in a bimer or oligomer or ahigher order aggregate through at least one component B.

Preferably at least two different bimer or oligomer types in accordancewith the present invention can also be contained in a composition foruse as a drug or as a vaccine, or for their production.

In a further embodiment in accordance with the present inventioncomponent A in a fusion protein in accordance with the present inventionis an immunomodulator, for example an interleukin (IL), in particularIL-2.

In addition, in the framework of the present invention the use of bimersor oligomers in accordance with the present invention as immunizationand/or vaccination adjuvans is disclosed. It is known that many antigensused for immunization trigger only an unsatisfactory immune reaction inthe test person. The task of adjuvans is to increase the immunogeniceffect. Adjuvans of this type can be used as component A in a fusionprotein in accordance with the present invention and therefore as acomponent of a bimer or oligomer in accordance with the presentinvention. For example, the component A can contain an amino acidsequence from the CD40 ligand (CD40L) or the sequence or sequencesection of an interleukin, for example one of the interleukins 1 to 12.The physiological task of CD40L is to control the transformation of aninactive B cell into the cell cycle. Interleukins, for example IL-4,IL-5, IL-6 and/or CD40L, can be combined in a bimerized or oligomerizedcomplex in accordance with the present invention (different recombinantfusion proteins in a bimer or oligomer), or they can occur as componentsof a composition (at least two different types of bimer or oligomer,which can each be developed from identical or different fusion proteins)which typically contains at least one, preferably two or more differenttypes of bimer or oligomer in accordance with the present invention,together with the immunogen(s).

The bimers of oligomers, or higher order aggregates as well, of acomposition of this type can be composed of fusion proteins which areidentical or different with regard to the component(s) A. Hereby, eachphysiological sequence with co-stimulating characteristics and/orcharacteristics which activate the immune system (cellular or humoralimmune response) can be considered as component A. These may bephysiological compounds or synthetic compounds. In this way,compositions are disclosed which contain one or more bimer or oligomertype(s) in accordance with the present invention together with one ormore immunogen(s), whereby a bimer or oligomer type in accordance withthe present invention can preferably be arranged so that more than oneimmodulator/immodulator adjuvans is contained in this type of bimerizedor oligomerized complex, in other words there is a heterobimer or aheterooligomer. If necessary, a heterobimer or a heterooligomer inaccordance with the present invention can bring together not only one ormore fusion proteins with an immunogen as component A, but also at leastone fusion protein with an adjuvans component as component A, forexample CD40L. Two or more different fusion proteins, each withdifferent adjuvans or immunomodulator components, are also conceivablein a heterooligomer in accordance with the present invention. This meansthat the invention also discloses the use as a drug or as a vaccine inhuman or veterinary medicine of these types of homoologimer orheterooligomer or of compounds which contain at least one type ofheterooligomer or homoologimer in accordance with the present invention.

In the framework of the present invention the bimers or oligomers areused preferably for the production of a drug or for the treatment of theabove-mentioned diseases or disorders in such a way that they aresuitable for parenteral administration, i.e., for example, subcutaneous,intramuscular or intravenous administration, or even for oral orintranasal administration. The administration of bimers or oligomers oraggregates of these as a vaccine or the basis for the production of avaccine, will also preferably take place in a parenteral or oral form ofadministration, but where necessary intranasal as well.

The bimers or oligomers in accordance with the present invention, and/orthe higher order aggregates, can be used alone as a medicament or can beincluded in the production of a medicament. However, they can also beused in combination with other active agent components as a medicament.The bimers or oligomers in accordance with the present invention, and/orthe higher order aggregates, can also be combined with pharmaceuticallyacceptable carriers, auxiliary agents or additives. Appropriateproduction paths are disclosed in Remington's Pharmaceutical Sciences(Mack. Pub. Co., Easton, Pa., 1980), which is part of the disclosure ofthe present invention. Examples of carrier materials which can beconsidered for parenteral administration are sterile water, sterile NaClsolutions, polyalkylene glycols, hydrogenated naphthalenes and inparticular biocompatible lactid polymers, lactid/glycolid copolymers orpolyoxyethylene/polyoxypropylene copolymers.

The bimers or oligomers in accordance with the present invention, orcorresponding higher order aggregates, are also used preferably in thefield of in-vitro diagnosis or, for example, for biochemical purifyingmethods. The use of bimers or oligomers and/or of higher orderaggregates of these on purifying columns, which can be packed with thesetypes of complexes, is to be considered. This means that in theframework of the present invention the use of these types of complexesis disclosed for the purposes of detection as well.

In addition, in the framework of the present invention processes aredisclosed to produce specifically associated proteins which interactionon the protein surface because of their interaction and as a result arefound dimerized or multimerized in solution. In particular with TNFcytokines, or preferably soluble segments of this type of cytokineswhich trimerize in solution, it is desirable to make them available in adefined stoichiometry in a pure fraction. In the case of a simplecoexpression of different proteins which are capable of associating withone another, for example of three different TNF cytokines or differentsegments of such TNF cytokines, all statistically possible distributionsof the coexpressed proteins are found in the trimers associated afterexpression, in other words, for example, the desired trimers from theproteins P1, P2 and P3, but also trimers from two proteins P1 and aprotein P3, etc.

Oligomers in accordance with the present invention can now be used inaccordance with the process in order to obtain defined desiredheteromultimers, for example, heterotrimers of TNF cytokines or segmentsof this type of TNF cytokines. For this purpose different fusionproteins are constructed and preferably expressed in a host cell. Thefusion proteins expressed here have a component B sequence sections ofproteins which form homoologimers from heteromultimers, for example,form a trimer out of three different chains, whereby identical trimersbimerise or oligomerise. Preferably, these components B in the fusionproteins correspond to sequence sections of proteins from the complementor collectin family, for example C1q, which forms homohexamers fromheterotrimers. In a fusion protein therefore, a sequence section, whichthe native protein provides for multimerizing a chain in theheteromultimer, is combined as component B with a component A, forexample a TNF cytokine, whereas other fusion proteins (which are tooccur in the heterotrimer) each have combinations of another componentA, for example a different TNF cytokine or a segment of this, withanother sequence section in the native protein, for example a C1qprotein, for heteromultimerizing.

The different fusion proteins which can form the heteromultimer areexpressed, preferably in a host cell. The heteromultimers combine intohomooligomers, because component B can only oligomerize identicalheteromultimers. In accordance with the present invention, for example,three different fusion proteins can be expressed, each with a differentcomponent A, in other words preferably different TNF cytokines, whicheach combine either with the multimerizing and oligomerizing α, β or γchain of C1q. Only heteromultimers with all three TNF cytokines can thenbe found in the associating oligomers. In contrast, heteromultimers witha different stoichiometry are not found. This means that in accordancewith the present invention simply through the selection of the fusionproteins a product can be obtained which is specific in itsstoichiometry and not subject to a statistical distribution.

In addition, the use is preferred of such fusion proteins or processesfor extracting heteromultimers with the given stoichiometry which have alinker between component A and component B. The linkers are speciallypreferred when they contain at least one proteolytic cleavage site whichpermits the components A from the homooligomer complex (ofheteromultimers) to be cleaved from the components B. In this way afraction is obtained which consists exclusively of the desiredheteromultimer, for example, a heterotrimer from the different TNFcytokines. The proteolytic cleavage site in the linker is preferably athrombin consensus sequence.

As a further object of the present invention fusion proteins aredescribed here which are suitable for bimerizing or oligomerizing dimersor multimers, in so far as the recombinant fusion protein contains atleast one component A and at least one component B, whereby thecomponent A contains a protein or a protein segment with a biologicalfunction, in particular with a ligand function for antibodies orreceptors or an antibody or segment of an antibody, and component Bcontains a dimerizing or multimerizing and bimerizing or oligomerizingsegment or a functional derivative of such a segment of a protein,selected from the group consisting of the family of C1q proteins or thecollectins. Extreme preference is given to these types of proteins ifthe component B of the recombinant fusion protein contains amultimerizing and/or an oligomerizing segment of the proteins C1q, SP-A,SP-D, BC, CL43 and ACRP30. A functional derivative of such a segment ofthe above-mentioned proteins can also be used in the framework of thepresent invention. In this case, together with component A havingbiological activity, a fusion protein will typically contain a componentB which has exclusively the segment of the above proteins which isresponsible far the aggregation, but preferably not the globular “head”domain thereof.

A sequence containing the amino acid sequence of the oligomerizingcollagen domain of the ligand EDA, in particular a mammalian variant,more particularly the human variant, or an oligomerizing fragment orfunctional derivate of such a domain is also considered as component Bof a fusion protein according to the present invention. Even morepreferred as component B, a sequence segment containing amino acids 160to 242 of the human EDA protein or a functional derivate, e.g. afragment, may be used. Preferably it may refer to a hexamer.

A further object of the present invention are DNA sequences which encodefor fusion proteins of the type referred to above. This type of DNAsequences is expressed in expression vectors, whereby the correspondingexpression vectors, which contain a DNA sequence for the fusion proteinsin accordance with the present invention, are also objects of thepresent invention.

In addition, host cells which are transfected with DNA sequences whichcode for the fusion proteins in accordance with the present inventionalso belong to the present invention. Extreme preference in this contextis given to host cells which are transfected with expression vectors,whereby the expression vectors again contain DNA sequences which codefor the fusion proteins in accordance with the present invention.

A further object of the present invention are receptors, in particularreceptors which bind signal molecule ligands from the TNF cytokinefamily which are found dimerized or multimerized. For example a receptorof this kind, a derivative of the same or a segment of a receptor or ofa derivative, in particular a segment which comprises the extracellularregion of the receptor, whereby once again the binding domain(s) is(are) preferred, can be found as a component A of a fusion protein whichis a component of a dimer or multimer. Dimerizing can be achievedthrough recombination with segments of immunoglobulins, in particular Fcfragments, whereas multimerizing can be achieved, for example, afterrecombination with the corresponding multimerizing domains of proteins.For this purpose, all sequence segments of proteins, for example, aresuitable which generate dimers or multimers through the formation ofsupersecondary structures, e.g. coiled-coil helices, or typicalcollagen-like triple helices (e.g. CMP, COMP, collagen, laminin).Segments of proteins from the C1q family or of collectins are alsotypically suitable for dimerizing or multimerizing receptors or receptorsegments. For example, the extracellular segment of a member of the TNFreceptor family, for example the Fas receptor (FasR) as component A inthe form of a pentamer, can be expressed as component B throughrecombination with the corresponding pentamerizing domains of COMP. Herethere may be homodimers, heterodimers or heteromultimers of fusionproteins which have a receptor or a receptor segment.

These dimers or multimers of a recombinant protein with a component Acontaining a receptor or a receptor segment may also be considered as amedicament or for the production of a medicament. Their use is inparticular given if increased extracellular concentrations of thecorresponding receptor ligands occur in a clinical picture. Here it mayalso concern increased concentrations of membrane-bound signalmolecules, for example TNF cytokines, on the cells themselves, or ofsoluble signal molecules. However, the use of multimers of this type isin principle also always desirable if the activation of a signaltransduction chain, which is triggered on the corresponding typicallymembrane-bound receptor, is to be prevented or lowered through the useof exogenous soluble dimers or multimers in accordance with the presentinvention which trap the signal molecules and which consist of fusionproteins which contain a receptor or a receptor segment as component A.The present invention is explained in detail by means of the followingfigures:

FIG. 1 shows in a single-letter code the amino acid sequence of arecombinant fusion protein in accordance with the present invention (2)occurring in an oligomer (FasL hexamer) in accordance with the presentinvention, in other words in the bimer of a trimer. A sequence segmentof the hFasL (AA 103 or 139 to 281) is identified as component A,whereas the specific linker (bimerizing in the outcome) with thesequence VDLEGSTSNGRQCAGIRL appears as component B. FIG. 1 also containsthe amino acid sequence of the recombinant fusion protein (1) which inaccordance with the state of the art only appears as component of a FasLtrimer, in other words does not have any component B which bimerizes oroligomerizes the existing multimer, in this case the trimer. Therecombinant segments of the two fusion proteins (1) and (2) are markedin FIG. 1 above the sequence details with regard to their respectivefunctionality.

FIG. 2 comprises in FIG. 2A the results of a gel electrophoresis(SDS-PAGE) of fusion proteins in accordance with the present invention(2), therefore with the specific linker sequence (component B), underreducing (r) and non-reducing conditions (nr). Under non-reducingconditions in solution the oligomer is eluted as a native complex of sixpolypeptides in accordance with the present invention (fusion proteins(2)), because in accordance with the present invention a disulphidebridge is formed between the components B of two fusion proteins (2) inaccordance with the present invention, which are each components ofdifferent trimers. The result is that there is an oligomer in accordancewith the present invention as a FasL hexamer with a molecular weightwhich appr. corresponds to six times the molecular weight of the fusionprotein (2) in accordance with the present invention in monomeric form.If denaturing conditions are present (e.g. on SDS-PAGE), the fusionproteins in accordance with the present invention migrate in the absenceof reducing agents as dimers, caused by the formation of a disulphidebridge between two monomers. In contrast, under reducing and denaturingconditions monomers of the fusion protein (2) in accordance with thepresent invention migrate in the SDS gel. An oligomer in accordance withthe present invention, here a bimer of a trimer, of a fusion protein (2)in accordance with the present invention, as shown in FIG. 1, is shownschematically in FIG. 2B.

FIG. 3 shows the results of the cytotoxic assay in dependence on theconcentrations of FasL trimers (trimers from three fusion proteins inaccordance with the state of the art, for example, fusion protein (1) inaccordance with FIG. 1 which do not have a component B), or of the FasLbimer (hexamer as bimer of trimers) in accordance with the presentinvention shown schematically in FIG. 2B in the presence (▪,

) or absence (□, Δ) of anti-flag M2 anti-bodies for A20 or Jurkat cells.The optical density at 490 nm is a measure of the viability of the cells(high optical density corresponds to a low apoptotic effect of the addedsubstances and thus a higher viability of the cells). The apoptoticeffect on A20 cells (FIGS. 3A and 3B) and on Jurkat cells (FIGS. 3C and3D) of FasL bimers of trimers (hexamers) in accordance with the presentinvention (FIGS. 3B and 3D, in each case Δ) increases 3 to 10 times asagainst the effect of FasL trimers of fusion proteins without abimerizing or oligomerizing component B (state of the art, FIGS. 3A and3C, in each case □). With additional anti-flag antibodies, which aredirected against the flag sequence of the fusion proteins (1) and (2)shown in FIG. 1 and by increasing, for example, the degree ofoligomerization of the fusion proteins in accordance with the presentinvention still further through cross-linking, the apoptotic effect isincreased in all preparations (FIGS. 3A to 3D, ▪ or

respectively).

FIG. 4 shows the amino acid sequence of a fusion protein (3) inaccordance with the present invention taking into account the (C→S)substitution in the specific linker section (component B of fusionprotein (3)) (“super FasL”). See the description of FIG. 1 otherwise.With gel-filtration experiments it was shown that “super FasL” insolution is found in a bimerized form in accordance with the presentinvention as a hexamer. Under denaturing conditions on SDS-PAGE, fusionproteins (3) in accordance with the present invention migrate, even inthe absence of reducing agents, as monomers, because a disulphide bridgecannot be formed in the linker section because of the amino acidsubstitution C→S.

Analogue to FIG. 3, FIG. 5 shows the viability of A20 (FIG. 5B) orJurkat cells (FIG. 5D) after the addition of aggregates in accordancewith the present invention of the fusion protein (3) in accordance withthe present invention as shown in FIG. 4 (“super FasL”) in the presence() or absence (∘) of anti-flag M2 antibodies. The fusion proteins (3)in accordance with the present invention bring about an apoptotic effectwhich is roughly at least 1000 times greater than the effect of FasLtrimers (used for control purposes) in accordance with the state of theart without bimerizing or oligomerizing components B (FIG. 3A (A20cells) and FIG. 3C (Jurkat cell's)) which, were used for comparison. Theaddition of anti-flag M2 antibodies () is able to increase theapoptotic activity both on A20 and on Jurkat cells slightly, approx.twice as much, preferably at least 1.5 times, as against the preparationwithout anti-flag antibodies, by further oligomerisation of “super FasL”to higher order aggregates in accordance with the present invention(FIGS. 5B and 5D). The result of this is that the degree ofoligomerisation (here as a bimer of trimers) for the apoptosistriggering, where necessary through oligomerisation on the cell surface,which is brought about through the specific linker of a fusion protein(3) in accordance with the present invention used as component B, isalready practically optimum and can be increased slightly by means ofhigher order aggregates in accordance with the present invention.

FIG. 6A shows the amino acid sequence of a fusion protein (4),FasL-ACRP30, in accordance with the present invention, whereby thefusion protein (4) (in the sequence from the N to the C terminus) has aflag sequence, a linker sequence as component B, the amino acids 18 to111 of protein mACRP30 (m: murine), the linker LQ and then the aminoacids 139 to 281 of hFasL as component A.

FIG. 6B shows a further fusion protein. It comprises the collagen domainof the human ACRP30 ligand (hACRP30, with amino acids 18 to 111) wherebythe N-terminus thereof is fused to a flag tag DYKDDDDK and theC-terminus thereof to the extracellular domain of human FasL (AA 139 to281). Between the C-terminus of hACRP30 and hFasL as well as between theFlag tag and the N-terminus of hACRP30 (GPGQVQLQ) a linker sequence(MHVD) is inserted. All above-mentioned data relate to theone-letter-code of amino acids.

FIG. 6C shows curves resulting from the titration of Jurkat T-cell withFasL on the one hand and with the fusion protein hACRP30/FasL on theother hand, under addition of anti-Flag antibody M2 (+) and withoutaddition of anti-Flag antibody M2 (−). Supernatants (OPTIMEM) of293-cells, being transiently transfected with FasL or hACRP30/FasL andexpressing these proteins, were therefore added to Jurkat T-cells. Insuccessive experiments decreasing concentrations shown on the x-axis ofFIG. 6C were employed and the respective viability rate of the JurkatT-cells was determined by the standard cytotoxicity test describedelsewhere. From the graph it is clear that FasL is inactive withoutcrosslinking M2 antibody (♦) and only the crosslinking effect of the M2antibody causes cytotoxic effects. In contrast thereto, thecorresponding effect of a fusion protein according to the presentinvention, namely hACRP30/FasL, shows already without addition of M2antibody (▴). The addition of M2 antibodies is hardly able to increasethe effect of the fusion protein according to the present invention.

Analogue to FIG. 3, FIG. 7 shows the viability of BJAB cells after theaddition of FasL trimers incapable of bimerizing or oligomerizing(comparative experiment on the basis of fusion protein (1) in accordancewith FIG. 1; FIG. 7A) and of oligomers in accordance with the presentinvention, here as tetramers of trimers, in other words dodekamers, ofthe fusion protein (4) in accordance with the invention and with FIG. 6in the presence (

) or absence (Δ) of anti-flag M2 antibodies. Whereas the FasL trimerswhich are not in accordance with the present invention do not develop anapoptotic effect on the BJAB cells in the absence of antibodies whicholigomerize through binding to the flag sequence (FIG. 7A, (Δ)), adodekamer in accordance with the present invention (tetramer of trimers)of fusion protein (4) induces cell death already at a concentration ofapprox. 10 ng/ml (K₅₀=8 ng/ml) (FIG. 7B). This can be seen quite clearlythrough a reduction of the OD at 490 nm. This apoptotic activity can beincreased slightly through the addition of anti-flag M2 antibodies to apreparation of dodekamers in accordance with the present inventionconsisting of FasL-ACRP30 fusion proteins in accordance with the presentinvention, i.e. a dodekamer in accordance with the present inventionrepresents a practically optimum aggregation status with regard to theapoptotic activity. In contrast, the further aggregation to higher orderaggregates in accordance with the present invention triggered bycorresponding antibodies is not able to bring about any furthersignificant biological effects.

The amino acid sequence of a fusion protein (5) in accordance with thepresent invention, which contains amino acids 95 to 281 of hTRAIL ascomponent A in combination with the oligomerizing domain of ACPR30 (AA18-111) as component B is shown in FIG. 8 (TRAIL-ACRP30). In accordancewith the present invention, therefore, the fusion protein (5) is foundin solution as an oligomer, namely as a tetramer of trimers.

FIG. 9 shows the viability of Jurkat cells after the addition of TRAILtrimers (fusion protein with a flag sequence at the N terminus and theamino acids 95 to 281 of human TRAIL) in accordance with the state ofthe art without the capability of bimerizing or oligomerizing(comparative experiment, FIG. 9A) and of dodekamers in accordance withthe present invention of the fusion protein (5) in accordance with thepresent invention, TRAIL-ACRP30, in the presence (

) or absence (Δ) of anti-flag M2 antibodies. The observations in theexperiment in FIG. 9 correspond to the findings shown in FIG. 7. Whereasthe TRAIL trimers do not develop an apoptotic effect on the Jurkat cellsin the absence of antibodies which oligoomerize through binding to theflag sequence (FIG. 9A, (Δ)), the dodekamer in accordance with thepresent invention of fusion protein (5) induces cell death in thisexperiment as well at a concentration of approx. 100 ng/ml (≈K₅₀) (FIG.9B (Δ)). By increasing the degree of oligomerization further, thecombined addition of TRAIL dodekamers and anti-flag antibodies can inaddition form higher order aggregates in accordance with the presentinvention, which in this case have an increased (at least tenfold)apoptosis-inducing activity (FIG. 9B, (

)), from oligomers in accordance with the present invention.

The amino acid sequence of a fusion protein (6) in accordance with thepresent invention, which contains the amino acids 77 to 235 of mTNFα (m:murine) as component A in combination with the oligomerizing domain ofmACRP30 (AA 18-111) as component B and a flag sequence with linker atthe N terminus, is shown in FIG. 10 (TNFα-ACRP30). This means that thefusion protein (6) is found in solution as a dodekamer, namely as anoligomer (tetramer) of multimers (trimers).

FIG. 11 shows the findings of a cell proliferation experiment. Here as avalue for the cell proliferation of CT6 cells the incorporation of ³[H]-thymidin into the CT6 cells of mice as a function of theconcentration of trimers of a fusion protein in accordance with thestate of the art (flag and linker sequence at the N terminus withsubsequent amino acids 77 to 235 from murine TNFα) without a component Bwas determined, in other words of the mTNFα trimer (FIG. 11A), or ofdodekamers (4×3) in accordance with the present invention of the fusionprotein (6) in accordance with the present invention according to FIG.6, mTNFα-ACRP30 (FIG. 11B). The incorporation of ³ [H]-thymidin is shownin counts-per-minute (cpm). The experiments were carried in the presence(

) or absence (Δ) of anti-flag M2 antibodies. Here the trimer of mTNFαwhich is familiar from the state of the art has only a slightproliferating effect on the CT6 cells after binding to the TNF receptor2 (TNF-R2) (FIG. 11A). A clearly increased proliferating effect can onlybe observed after the addition of anti-flag antibodies (

) through their cross-linking and therefore oligomerizing effect.

In contrast to this, in FIG. 11B oligomers in accordance with thepresent invention of the trimers, here the dodekamer of the fusionprotein (6) in accordance with the present invention, show a heavyproliferating effect which can already be observed at 5 ng/ml (Δ). Thecombination of cross-linking anti-flag antibodies and dodekamers (

) in accordance with the present invention as reference can in contrastonly bring about a slight increase of the proliferating effect incomparison with the findings with the sole addition of the dodekamer inaccordance with the present invention. It can be seen from this that anoligomer in accordance with the present invention, here in form of adodekamer, already has a practically optimum proliferation effect.

The amino acid sequence of a fusion protein (7) in accordance with thepresent invention, which contains the amino acids 116 to 261 of hCD40L(h: human) as component A in combination with the oligomerizing domainof ACRP30 (AA 18-111) as component B, is shown in FIG. 12(CD40L-ACRP30).

FIG. 13 shows, in exactly the same way as FIG. 11, the findings of acell proliferation experiment. However, in this case, in contrast toFIG. 11, human PBL (peripheral blood lymphocytes) were used. The figureshows the effect of traditional CD40L, which is present in solution inthe form of a trimer (flag and linker sequences, as in FIG. 12, withsubsequent sequence from AA 16 to 261 of hCD40L without an oligomerizingcomponent B; FIG. 13A), on the cell proliferation in comparison with theeffect of dodekamers in accordance with the present invention of fusionproteins (7) in accordance with the present invention as shown in FIG.12, CD40L-ACRP30 (FIG. 13B). The experiments carried out in the absence(Δ) of anti-flag M2 antibodies show that the CD40L trimer haspractically no proliferating effect, whereas in FIG. 13B a correspondingproliferating effect can already be detected with much lowerconcentrations of hCD40L. In FIG. 13B, the x-axis shows the variable“1/dilution”, in other words, not absolute concentrations, because aconcentrated supernatant, whose absolute concentration is unknown, wasadded. The combination of cross-linking anti-flag antibodies anddodekamers of fusion protein (7) in accordance with the presentinvention (

) as reference can generate a further (to lower concentrations) shift ofa corresponding proliferation effect through the formation of higherorder aggregates.

FIG. 14 shows a schematic representation of the structure ofoligomerized multimers. FIGS. 14A and 14B show schemas of the appearanceof native “bundle proteins” with their already native oligomerizedstructure (FIG. 14A: hexamer of head domain trimers which form thecomplement protein C1q; FIG. 14B: tetramer of head domain trimers whichform ACRP30, a serum protein produced through adipocytes). In this way,the head domains of the native monomers at the C1q or ACRP30 complex aremultimerized and oligomerized. Whereas the C1q oligomerizing complex isformed from three different gene products, the ACRP30 oligomerizingcomplex is a homododekamer, in other words identical multimerized andoligomerized gene products. The respective individual polypeptide chainson which the above-mentioned native oligomerizing complexes are basedeach have a head domain, a sequence section which can form a collagentriple helix, and a sequence section which can oligomerize 6 (C1qcomplex) or 4 (ACRP30 complex) collagen triple helices into a helixbundle of 18 or 12 monomers, respectively.

FIG. 14C contains a schematic representation of a oligomerized multimerin accordance with the present invention of a fusion protein inaccordance with the present invention (e.g. fusion protein (4) as shownin FIG. 6, FasL-ACRP30), which has four trimerized TNF ligands (e.g. theTNF ligand FasL) or segments of TNF ligands as component A, whicholigomerize to a homododekamer in accordance with the present inventionthrough the collagen-like sequence sections of the ACRP30 protein ascomponent B, which is found, for example, in a fusion protein inaccordance with the present invention.

FIG. 15A describes the construction of a further fusion proteinaccording to the present invention, namely hEDA/FasL. Herein, thecollagen domain of human EDA (amino acids 160 to 242) serves ascomponent B to which a Flag epitope is fused N-terminally via a linkerand to which a component A, FasL (AA139-281, i.e. the extracellulardomain of FasL or a fragment thereof) in FIG. 15A, is coupledC-terminally also via a linker. The fusion protein hEDA/FasL accordingto the present invention is present as a hexamer, a 2×3 mer. The EDAprotein is a further member of the TNF family, which has also a collagendomain (FIG. 15A above) besides a transmembrane domain and a TNF domain,and comprises 391 AA in its human form.

FIG. 15B illustrates studies with the fusion protein hEDA/FasL accordingto the present invention. For the production of the fusion protein, thecollagen domain of human EDA (AA 160 to 242) was initially amplified bycorresponding primers and then fused to the corresponding sequences atthe N- and C-terminus, respectively, for Flag and the extracellulardomain of human FasL (AA 139 to 281). Now, FIG. 15B represents curvesresulting from the titration of Jurkat T-cells with FasL on the one handand the fusion protein hEDA/FasL on the other hand, under addition ofanti-Flag antibody M2 and without addition of anti-Flag antibody M2.Supernatants (OPTIMEM) of 293-cells, being transiently transfected withFasL or hEDA/FasL and expressing these proteins, were therefore added toJurkat T-cells. In successive experiments, the decreasing concentrationsshown on the x-axis of FIG. 15B were employed and the respectiveviability rate of the Jurkat T-cells was determined by the standardcytotoxicity test described elsewhere. From the graph it is clear thatFasL is inactive without crosslinking M2 antibody (□), and in this caseonly the crosslinking effect by the antibody M2 causes cytoxic effects(▪). In contrast thereto, the corresponding effect of a fusion proteinaccording to the present invention, namely hEDA/FasL, is alreadyachieved without addition of M2 antibody (∘). In this case, the additionof M2 antibodies is still able to increase the effect of the fusionprotein according to the present invention ().

The present invention is explained in more detail by means of thefollowing embodiments:

The following experimental situations (a) to (f) are to be referred tofor the six following embodiments, in so far as appropriate andcorresponding modifications disclosed loc. cit. do not apply:

-   -   (a) Vector Constructions for the FasL, TRAIL, TNFα and CD40L        fusion proteins

A DNA fragment encoding for the signal peptide of haemagglutinin,including 6 bases of the untranslated sequence in the 5′ region (CAA AACATG GCT ATC ATC TAC CTC ATC CTC CTG TTC ACC GCT GTG CGG GGC) and theflag epitop (GAT TAC AAA GAC GAT GAC GAT AAA), the linker (GGA CCC GGACAG GTG CAG), the restriction sites PstI, SalI, XhoI and BamHI werecloned between the restriction sites HindII and BamHI of a modifiedPCRIII vector (InVitrogen, NV Leek, Netherlands) in which the bases720-769 were deleted (PS 038). For the expressions vector of thetrimeric FasL the amino acids 139 to 281 of the sequence encoding forhuman FasL, framed by the restriction sites PstI and EcoRI, wereamplified with PCR and cloned in the modified PCRIII vector.

For the hexameric FasL the encoding sequence for the amino acids 103 to281, framed on both sides by EcoRI restriction sites and in addition atthe 5′ end by the linker sequence GGCTT and at the 3′ end by the stopcodon (TAA) and the natural, untranslated sequence (GAG AAG CAC TTT GGGATT CTT TCC ATT ATG ATT CTT TGT TAC AGG CAC CGA GAT GTT GAA GCC) wascloned into the EcoRI restriction site of the vector PS 038. The “superFasL” (FIG. 4, fusion protein (3)) was generated by the introduction ofa point mutation in the linker sequence of the vector PS 038, in thatthe sequence CAGTGTGCTG on the 5′ side of the EcoRI restriction site wasreplaced by CAGTCTGCAG with the help of PCR mutation methods. Followingthis the FasL (amino acids 103 to 281) was cloned into the modified PS038 in the manner described above.

For human TRAIL, murine TNFα and human CD40L, parts of the extracellulardomains (TRAIL: amino acids 95 to 281, TNFα: amino acids 77 to 235,CD40L: amino acids 116 to 261) with PstI restriction sites at the 5′ endand a stop codon and SpeI and EcoRI restriction sites were amplifiedthrough PCR and cloned in the vector PCRII (Invitrogen). For theexpression of the ligands as trimers firstly the sequence GAT TAC AAAGAC GAT GAC GAT AAA, encoding for the flag tag, and the linker sequenceGGA CCC GGA CAG GTG CAG were inserted between the restriction sitesBamHI and PstI in the vector pQE-16 (Qiagen) (PS 330). Finally, theligands were sub-cloned as PstI/SpeI fragments in PS 330.

The expression vector for FasL-ACRP30 was constructed in the followingway. By means of the EST clone AA673154, the encoding sequence for theamino acids 18 to 111 of the murine ACRP30, framed by the restrictionsites NsiI and PstI, was cloned by PCR methods into the PstI restrictionsite of the vector encoding for trimeric FasL (in such a way that thefusioned NsiI/PstI restriction site was located on the 5′ side of theencoding sequence). The vectors for the expression of the fusionproteins of the other TNF cytokines with ACRP30 were created bysubstitution of the corresponding sequence of FasL in the expressionvector FasL-ACRP30 by the respective ligand sequence into therestriction sites PstI and EcoRI.

-   -   (b) Expressing and Purifying the Recombinant Fusion Proteins:

Stable clones were established in bacteria (strain M15 with plasmidpRep4, Qiagen) for the trimeric TRAIL, TNFα and CD40L. The respectiveclones were precultivated overnight at 37° C. in LB broth withampicillin (100 μg/ml) and kanamycin (50 μg/ml) and were used toinoculate the main culture (dilution 1:50, growth at 37° C.), which wasinduced for the expression after one hour with 0.5 mM IPTG (Sigma) forsix hours. The bacteria were harvested by means of centrifugation,washed twice in PBS, lysated in the “French press” and the lysate wasseparated from the insoluble rest by centrifugation. Stable clones weremade for all FasL proteins in HEK293 cells by means of selection in 800μg/ml G418 (see loc. cit. as well: Schneider et al., J. Exp. Med, 1998).

The trimeric and hexameric ligands and super FasL were purified from thesupernatants of stable clones or of bacterial lysates by means ofaffinity chromatography on M2 agarose (Sigma, Switzerland), eluted with50 mM citrate NaOH (pH=2.5) and immediately neutralized with 0.2 volumeTris-HCl (pH=8). The buffer was replaced by PBS in concentrators(Centrikon-30, Amicon Corp., Easton, Tex., USA).

Fusions of FasL with murine ACRP30 were purified in the followingmanner. The supernatants were mixed with 50 mM NaCl and 1 mM CaCl₂ andthe pH value was set to 7.0 by means of hydrochloric acid/NaOH. Therecombinant protein was then bound to M1-agarose (Sigma, Switzerland)and eluted in TBS-EDTA (10 mM). The buffer was replaced by PBS inconcentrators. The concentration of purified proteins was determinedthrough the bicinchonin acid process (Pierce Chemical Co., Rockford,Ill., USA) using bovine serum albumin as a standard and the purity ofthe samples was determined through SDS-PAGE and Coomassie Blue staining.

The fusion proteins of TRAIL, TNFα and CD40L with ACRP30 were added inthe respective assays in the form of enriched supernatants which wereproduced in the following manner: 293T cells were transfected with therespective expressions plasmids through the calcium phosphate method.After the cells had been incubated with the precipitate for 16 hours,the cells were washed with PBS and incubated for 4 days in a serum-freemedium (Optimen, Gibco BRL). The supernatants were reduced toone-twentieth of the original volume through concentrators and thepresence of the protein was checked with Western blotting. In the caseof TRAIL-ACRP30 and TNFα-ACRP30 titrations of these proteins werecompared with titrations of purified trimeric TRAIL or TNFα in order todetermine the respective concentration of the recombinant fusionproteins.

-   -   (c) Checking the Molecular Weight of the Multimers Through        Gel-Permeation Chromatography:

The size of the different fusion proteins was determined throughgel-filtration on Superdex 200 HR10/30 (Pharmacia). Recombinant proteinwith the internal standards catalase and ovalbumin in the case of thetrimeric and hexameric ligands, and ferritin and ovalbumin for theACRP30 fusion proteins, was loaded onto the column in a volume of 200μl, eluted with PBS (0.5 ml/min) and collected in fractions of 0.25 ml.The presence of the proteins in the different fractions afterprecipitation through trichloro ethanoic acid was determined withWestern blotting. The size of the proteins was determined with the helpof thyroglobulin (669 kDa), ferritin (440 kDa), catalase (262 kDa),aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa)chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).

-   -   (d) Cells:

Murine B-lymphoma A20 cells were held in DMEM which contained 5%heat-inactivated FCS. The human T-lymphoplastom-Jurkat cells, BJABBurkitt's-lymphoma cells were cultivated in RPMI, accompanied by 10%FCS. The human embryonic renal cells 293 were cultivated in a DMEMmulti-material mixture F12 (1:1), accompanied by 2% FCS. All mediacontained antibiotics (penicillin and streptomycin at 5 μg/mlrespectively and neomycin at 10 μg/ml). The IL-2 dependent murinecytoxic T-cell line CT6 was cultivated in RPMI, supplemented by 10% FCS,1 mM natriumpyruvate, 50 μM 2-mercaptoethanol, 10 mM hepes and 10%conditioned EL-4 cell supernatant.

-   -   (e) Cytotoxic Assay:

The cytotoxic assay was carried out essentially as described previouslyby Schneider et al. (J. Biol. Chem., 272:18827-18833, 1997). Fiftythousand cells were incubated for a period of 16 hours in 100 μl medium,whereby the medium contained the displayed ligand concentrations in thepresence or absence of 1 μg/ml M2 antibody (2 μg/ml for TRAIL). Theviability (cell survival rates) were determined with the help of PMS/MTS(phenanzinmethosulphate3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium,salt] (Promega Corp., Madison, Wis.). The colour development was allowedfor the required time (typically 1-3 hours). The absorbance was measuredat 490 nm.

-   -   (f) B Cell Proliferation Assay:

CD19 positive cells were selected by means of FACS sorting from humanPBL (peripheral blood lymphocytes) with magnetic “beads” and purified,whereas CD-19 negative cells were irradiated with 3000 rad. One hundredthousand purified CD-19 positive cells were incubated for 72 hours in96-well plates with 100,000 autologous CD-19 negative irradiated PBL in120 μl medium (RPMI 10% FCS, antibiotics) with the titrated solubleCD40L fusion proteins, with or without M2 antibodies (10 μg/ml).Subsequently, the cells were pulsed for 6 hours with [³H]-thymidin andthe incorporation was measured with liquid scintillation counting.

With regard to the description of the methods used for implementing theembodiments explicit reference is made otherwise to Schneider et al. (J.Exp. Med., Vol. 187, No. 8, 1998, 1205-1213) and the publications quotedthere as references.

1ST EMBODIMENT

A recombinant fusion protein (2) was expressed which had the amino acids103 to 281 of the hFasL (h: human) as component A and at the N-terminusof amino acid 103 a sequence of 18 AA (VDLEGSTSNGRQCAGIRL, so-calledspecific linker) as component B. In addition, a flag sequence with theamino acids DYKDDDDK and a linker sequence GPGQVQLQ following this wascoupled at the N-terminus of the fusion protein (N-terminal of componentB) (FIG. 1).

For comparative experiments, a fusion protein (1) was expressed whichalso had the above-mentioned flag sequence at the N-terminus with thesame linker sequence following at the C-terminus and connected to thisat the C-terminus the amino acids 139 to 281 of hFasL. Accordingly,fusion protein (1) differs from fusion protein (2) through a deletionwhich covers the specific linker and the amino acids 103 to 138 of hFasL(FIG. 1).

The vector construction of the fusion proteins (1) and (2) took place inaccordance with the procedure described in (a). The expression andpurification of the fusion proteins took place in accordance with theprocedure described in (b).

The purified fusion proteins (1) were subjected to reducing ornon-reducing conditions and then gel electrophoretically separated (FIG.2), linked with a rough determination of the molecular weight of therespective bands.

Finally, A20 and Jurkat cells cultivated in accordance with (d) wereremoved and subjected to a cytotoxic assay in accordance with (e). Theassay was carried out (FIG. 3) for each of the two cell lines withincreasing concentrations of trimerized fusion protein (1) or of bimersof trimers (in other words hexamers) of the fusion protein (2) in thepresence or absence of anti-flag M2 antibodies (Sigma, Buchs,Switzerland), in that the absorbance was determined at OD 490 mm.

2^(ND) EMBODIMENT

A recombinant fusion protein (3) was expressed which had the amino acids103 to 281 of the hFasL as component A and at the N-terminus of aminoacid 103 a sequence of 18 AA (VDLEGSTSNGRQSAGIRL, so-called specificlinker) as component B. In addition, a flag sequence with the aminoacids DYKDDDDK and a linker sequence GPGQVQLQ following this was coupledat the N-terminus of the fusion protein (N-terminally from component B),(FIG. 4).

For comparative experiments the fusion protein (1) was expressed in thesame way as the 1^(st) embodiment (FIG. 4).

The vector construction of the fusion proteins (3) and (1) took place inaccordance with the procedure described for embodiment 1. The expressionand purification of the fusion proteins took place in accordance withthe procedure described in (b).

Finally, A20 and Jurkat cells cultivated in accordance with (d) wereremoved and subjected to a cytotoxic assay in accordance with (e). Theassay was carried out (FIG. 5) for each of the two cell lines withincreasing concentrations of trimerized fusion protein (3) in thepresence or absence of anti-flag M2 antibodies (Sigma, Buchs,Switzerland), in that the absorbance was determined at OD 490 mm.

3^(RD) EMBODIMENT

A recombinant fusion protein (4) was expressed which had the amino acids139 to 281 of the hFasL as component A and at the N-terminus of aminoacid 139 of component A firstly the linker dimer with the sequence LQand then still further N-terminally a sequence of 94 AA from the proteinmACRP30 (AA 18 to 111), the oligomerizing domain, as component B. Inaddition, a flag sequence with the amino acids DYKDDDDK and a linkersequence GPGQVQLH following this was coupled at the N-terminus of thefusion protein (4) (N-terminally from component B) (FIG. 6).

The fusion protein (1) was used for comparative experiments.

The expression and purification of the fusion proteins took place inaccordance with the procedure described in (b).

Finally, BJAB Burkitt's lymphoma cells and Jurkat cells cultivated inaccordance with (d) were removed and subjected to a cytotoxic assay inaccordance with (e). The assay was carried out (FIG. 7) for each of thetwo cell lines with increasing concentrations of trimers of the fusionprotein (1) or of oligomerized trimers (dodekamers) of fusion protein(4), that means of the recombinant FasL-ACRP30 (4×3), in the presence orabsence of anti-flag M2 antibodies (Sigma, see above), in that theabsorbance was determined at OD 490 mm.

4^(TH) EMBODIMENT

A recombinant fusion protein (5) was expressed which had the amino acids95 to 281 of the hTRAIL (h: human) as component A and at the N-terminusof amino acid 95 of component A firstly the linker dimer with thesequence LQ and then more N-terminally a sequence of 94 AA from theprotein mACRP30 (AA 18 to 111), the oligomerizing domain, as componentB. In addition, a flag sequence with the amino acids DYKDDDDK and alinker sequence GPGQVQLH following this was coupled at the N-terminus ofthe fusion protein (N-terminally from component B) (FIG. 8).

For comparative experiments a fusion protein was expressed (not shown inFIG. 8) which is found in solution as a trimer (TRAIL trimer). Thiscomparative experiment protein has (from the N-terminus to theC-terminus) the flag sequence, the linker with the sequence GPGQVQLH andfinally hTRAIL (AA 95 to 281). In contrast to fusion protein (5) thecomponent B (mACRP30: AA 18 to 111) and the linker with the sequence LQare missing.

The expression and purification of the fusion proteins took place inaccordance with the procedure described in (b).

Finally, T-lymphoblastoma Jurkat cells cultivated in accordance with (d)were removed and subjected to a cytotoxic assay in accordance with (e).The assay was carried out (FIG. 9) with increasing concentrations oftrimers of the fusion protein without the ACRP30 sequence (for acomparison) or of the oligomerized trimers of the fusion protein (5), inother words of a dodekamer of the recombinant TRAIL-ACRP30 (4×3), in thepresence or absence of anti-flag M2 antibodies (Sigma, see above), inthat the absorbance was determined at OD 490 mm.

5^(TH) EMBODIMENT

A recombinant fusion protein (6) was expressed which had the amino acids77 to 235 of the mTNFα (m: murine) as component A and at the N-terminusof amino acid 85 firstly the linker dimer with the sequence LQ and thenfurther N-terminally a sequence of 94 AA from the protein mACRP30 (AA 18to 111), the oligomerizing domain, as component B. In addition, a flagsequence with the amino acids DYKDDDDK and a linker sequence GPGQVQLHfollowing this was coupled at the N-terminus of the fusion protein(N-terminally from component B) (FIG. 10).

For comparative experiments a fusion protein was expressed (not shown inFIG. 10) which is found in solution as a trimer (TNFα trimer). Thiscomparative experiment protein has (from the N-terminus to theC-terminus) the flag sequence, the linker with the sequence GPGQVQLH andfinally mTNFα (AA 77 to 235). In contrast to fusion protein (6) thecomponent B (mACRP30: AA 18 to 111) and the linker with the sequence LQare missing.

The expression and purification of the fusion proteins took place inaccordance with the procedure described in (b).

With the help of a cell proliferation assay in accordance with (f) theeffects of adding increasing concentrations of TNFα trimers orTNFα-ACRP30 oligomers (homododekamers of TNFα) in the presence orabsence of anti-flag M2 antibodies (Sigma, see above) to CT6 cells weredetermined.

For this purpose, CT6 cells were prepared for a period of 4 days beforethe proliferation experiment in the presence of reduced concentrationsof EL-4 supernatants (2.5%). The cells were incubated in 96-well titreplates (40,000 cells per well) for a period of 16 hours with theindicated concentrations of TNFα-ACRP30 oligomers or mTNFα in thepresence or in the absence of 2 μg/ml M2 monoclonal antibodies and inthe absence of EL-4 supernatant. The cells were pulsed for an additionalperiod of 6 hours with ³[H]-thymidin (0.5 μCi/well), subjected to threecycles of freezing and thawing and finally harvested. The ³[H]-thymidinincorporation was finally checked by means of a liquid scintillationmethod (FIG. 11).

6^(TH) EMBODIMENT

A recombinant fusion protein (7) was expressed which had the amino acids116 to 261 of the hCD40L (h: human) as component A and at the N-terminusof amino acid 95 of component A firstly the linker dimer LQ and thenfurther N-terminally a sequence of 94 AA from the protein mACRP30 (AA 18to 111), the oligomerizing domain, as component B. In addition, a flagsequence with the amino-acids DYKDDDDK and a linker sequence GPGQVQLHfollowing this was coupled at the N-terminus of the fusion protein(N-terminally from component B) (FIG. 12).

For comparative experiments a fusion protein was expressed (not shown inFIG. 12) which is found in solution as a trimer (CD40L trimer). Thiscomparative experiment protein has (from the N-terminus to theC-terminus) the flag sequence, the linker with the sequence GPGQVQLH andfinally hCD40L (AA 116 to 261). In contrast to fusion protein (7) thecomponent B (mACRP30: AA 18 to 111) and the linker with the sequence LQare missing.

The expression and purification of the fusion proteins took place inaccordance with the procedure described in (b).

Finally, the cell proliferation assay in accordance with (f) was carriedout analogously on PBL, whereby CD40L trimer CD40L-ACRP30 oligomers(homododekamers) were added in the presence or absence of anti-flag M2antibodies (Sigma, see above) (FIG. 13).

7^(TH) EMBODIMENT 7.1 Experimental Procedures

The 7^(th) embodiment refers to fusion proteins which consists of amultimerizing and oligomerizing component A and a receptor as componentB.

(A) Vector Constructions

The fusion proteins were constructed from a modified PCR-3 Vector (fromInvitrogen) as an arrangement of interchangeable modules in thefollowing order (5′ to 3′):

(a) a HindIII/SalI modul, containing the extracellular domain ofreceptor, with a preceeding Kozak sequence GCCACC (in the case ofhTNF-R1 (amino acids 1-211); h-TRAIL-R1 (amino acids 1-239); h-TRAIL-R2(amino acids 1-212); h-TRAIL-R3 (amino acids 1-240) and hCD40 (aminoacids 1-193) or in the case of hFasR (amino acids 1-170) by placing infront 24 nucleotides of the 5′-untranslated region); (b) a 14 aminoacid-long linker (PQPQPKPQPKPEPE) within a SalI/XhoI-cassette (asdescribed in Terskikh et al. (1997), Proc. Natl. Acad. Sci. USA 94,1663); (c) an oligomerization domain in an XhoI/NotI-module in the caseof OPG (amino acids 187-401, herein designated as δN-OPG), CMP (aminoacids 451-493), GenBank 115555); COMP (amino acids 32-75, GenBank1705995); (d) a NotI/XbaI-cassette, containing a combined His₆-myc-tagand a stop codon. The oligomerization domain was framed by the aminoacid sequences GGCC and ARTPGGGS at the N- and C-termini, respectively.Linkers were used for all constructs. In the case of Fc-constructs the“hinge” region, the CH2 and CH3 domains and the stopcodon of hIgG1 werecloned as SalI/NotI-cassette as described before (Schneider et al.(1997) J. Biol. Chem. 272, 18827; Schneider et al. (1998) J. Exp. Med.187, 1205). Stable HEK-293 derived cell lines for the production ofrecombinant proteins were established by selection in 800 μg/ml G418 bythe method described previously (Schneider et al. (1997), loc. cit.).

(B) Transient Transfection

293T cells were transfected by the CaCl₂ method as described before(Schneider et al. 1997, loc. cit.) and washed with PBS before they wereincubated in serum-free Optimem medium for 3 days. Supernatants wereconcentrated 30 fold and maintained frozen until needed. Theconcentration of Fas/δN-OPG and Fas/CMP fusion proteins in concentratedOptimem medium was determined by a titration on “western blots” with theaid of purified Fas/COMP as standard.

(C) Purification of Recombinant Proteins

Supernatants of stably transfacted 293-cells were loaded on M2-agarose(as ligand) or protein A-sepharose (Fc fusion proteins), washed with PBSand eluted by 50 mM citrate-NaOH (pH 2.5). The eluate was neutralized byTris-HCl (pH 8) and the buffer was changed to PBS in centrikon30concentrators (from Amicon, Easton, Tex.). COMP and CMP fusion proteinswere purified on HiTrap-chelate columns. For this purpose, supernatantsof stabily transfacted 293 cells were supplemented by 500 mM NaCl and 10mM imidazol, and given on columns which were coated with 0.5 M ZnSO₄ (pH2.5), and equilibrated in PBS. The column was washed with PBS and theproteins were eluted with PBS containing 50 mM EDTA. The buffer waschanged to PBS as described before.

Flag-FasL and Flag-TRAIL were produced as described before (Schneider etal., 1997, J. Biol. Chem. 272, 18827, and Thome et al., 1997, Nature386, 517). Both references are included in the disclosure of thesubject-matter of the present application by reference. Flag-CD40L (AA116 to 261) was expressed in bacteria and purified on M2-agarose, asdescribed for Flag-TRAIL. Protein concentrations were determined by thebicinchonic acid method. The degree of purification was examined bySDS-PAGE and Coomassie-Blue staining.

(D) Gel Permeation Chromatography

For gel permeation chromatography, the respective amount of fusionprotein in a volume of 200 μl was loaded on to a Superdex 200 HR 10/30column (from Pharmacia) and eluted in PBS by 0.5 ml/min, concomittantlymeasuring the absorbance at 280 nm. As described below, the individualfractions (0.25 ml) were analysed in cytotoxicity tests. For thedetermination of the molecular weight, the column was calibrated withthe standard proteins thyroglobulin (669 kD), ferritin (440 kD),catalase (262 kD), aldolase (158 kD), bovine serum albumin (67 kD),chicken ovalbumin (43 kD), chymotrypsinogen A (25 kD) and ribonuclease A(13.7 kD).

(E) Competitive ELISA

The competitive ELISA was carried out as follows. 96 “well”-ELISA-plateswere coated with receptor/Fc-fusion protein (0.2 μg/ml in PBS, 100 μl,16 hours, 25° C.). The wells were saturated for one hour at 37° C. inPBS, the PBS containing 5% fetal calf serum (as blocking buffer). Forthe use of sCD40L the blocking buffer was PBS, containing 4% fat freemilk and 0.05% tween 20. Competing Fc or COMP fusion proteins wereserially dissolved in 100 μl blocking buffer in the presence or absenceof 1 μg/ml protein A. The ligands were added at a constant concentration(sFasL: 2 μg/ml, 36 nM, sTNFα: 0.02 μg/ml, 0.36 nM; sTRAIL: 0.1 μg/ml1.4 nM; sCD40L: 0.5 μg/ml, 9.2 nM all in blocking buffer) and wereallowed to bind for a period of one hour at 37° C. Bound ligands wereidentified with an M2-anti-Flagantibody (1 μg/ml in blocking buffer, 100μl, 45 minutes, 37° C.), peroxidase-conjugated goat-anti-mouseantibody,1:2000 in blocking buffer, 100 μl, 30 minutes, 37° C.) andophenylenediamine hydrochlorid (0.3 mg/ml in 50 mM citric acid, 100 mMNa₂HPO₄, 0.01% H₂O₂). The absorbance was measured at 490 nm.

(F) Cytotoxicity Tests

The cytotoxicity tests were carried out in 96-well plates in a volume of100 μl, substantially as described in Schneider et al. (1997), loc. cit.The chimeric receptors were serially diluted in a medium containingamounts of cytotoxic ligands, which were able to induce more than 95% ofcell death. Where indicated, protein A was used at a concentration of 1μg/ml. sFasL was used in the presence of 1 μg/ml M2 and sTRAIL in thepresence of 2 μg/ml M2. No M2-antibody was used in the test series withsTNFα. The cells were exposed to a 16 hour incubation, and theirviability rates were measured by the use of the PMS/MTS-test systems(phenacinemethosulfate/3-[4,5-dimethylthiazole-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium,in form of a salt) (Promega, Madison, Wis.). The absorbance was measuredat 490 nm. In order to indicate the molar concentrations of the variousfusion proteins, the molecular weight was estimated as follows:[(theoretical M_(r)+3 kDa per predicated N-coupled glyan)×multiplicity].Fas:Fc, :COMP, :CMP, :δN-OPG: 98, 172, 100 and 105 kDa, respectively.TRAILR2:Fc, :COMP:, :CMP, :δN-OPG: 86, 142, 82 and 93 kDa. TNFR1:Fc,:COMP: 104 and 187 kDA. CD40:Fc, :COMP: 101 and 180 kDa. TRAILR2:Fc: 86kDA, TRAILR3:Fc: 127 kDA. Flag-TRAIL, Flag-FasL, Flag-TNFα, Flag-CD40L:71, 55, 56 and 54 kDa, respectively.

(G) BIAcore Measurements

The biacore measurements were carried out onCM5-carboxymethyldextran-modified sensor chips (from BIAcore AB,Uppsala, Sweden) at a flow rate of 5 μl/min. The CM5-chips wereactivated by a 50 μl dose of a 1:1 mixture ofN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide:N-hydroxysuccinimide.Then, six μl of a 100 μg/ml solution of M2-anti-Flag monoclonal antibodyin 10 mM NaHCO₃ (pH 5.5) were delivered over the activated surface. A 50μl dose of 1M ethanolamine-HCl deactivated the remainingN-hydroxysuccinimidester. The amount of immobilised M2-antibody, whichas needed for the procedure, was about 4600 units. For the analysis ofinteractions of FasL-Fas:fusion protein and TRAIL-TRAIL-R2:fusionprotein, a constant amount of labelled ligand was immobilised on anM2-modified surface. In order to achieve this, a 7 μl dose of 2.5 μg/mlFlag-FasL or Flag-TRAIL was brought on the surface. This led to thebinding of about 100 to 150 units. Besides, these conditions allowed aminimal dissociation of the ligands from the M2 surface, as theyconcomittantly allowed a sufficient subsequent receptor binding for theanalysis. The binding of the receptor:fusion protein was then analysedby a 15 μl injection of purified receptor:fusion protein atconcentrations between 1 and 100 μg/ml, corresponding to 100 to 150units. The association kinetics were measured for a period of 3 minutesand the dissociations kinetics for a period of 3 to 5 minutes. Then, thesurface was regenerated (to a simple M2 surface) by a 5 μl dose of 50 mMcitrate-HCl (pH 2.5). Up to 30 successive repeats of binding andregeneration were carried out without a significant change in thebinding characteristics of immobilised M2-antibody. The dissociation andassociation kinetics were analysed with the aid of the kinetic analysisprogram provided by the manufacturer using the models AB=A+B and A+B=AB.

(H) Cultivation of Primary Mice Hepatocytes

For the cultiviation of primary mice hepatocytes, C57BL/6 mice weresacrificed, and the liver region above the biliary vessel was removedimmediately, in order to then maintain it in a “hepatocyte-attachmentmedium” (HAM). Initially, the liver region was perfused with 16 ml of 10mM Hepes (pH 7.6) in order to remove erythrocytes, then with 12 ml of0.5 mg/ml collagenase H (from Boehringer Mannheim) in Hepes (4 mMCaCl₂), and then homogenised in Hepes in a Petri dish. The cells werewashed in Hepes, then centrifuged (100 xg, 30 seconds) and resuspendedin 60% isotonic. Percoll solution (from Pharmacia) in HAM and againcentrifuged (700 xg, 2 min). All buffers and the medium were used at 37°C. The sedimented cells were resuspended in HAM, counted, seeded onflatdish-formed microtiterplates (10000 per well, 200 μl) and wereallowed to attach correspondingly. The experimental mixture (seriallydiluted inhibitors, sFasL (final concentration: 400 ng/ml, 7 nM) andM2-antibody (final concentration: 1 μg/ml)) were added, and then thecells were incubated for further 16 hours. The Supernatant was removed,fresh HAM (100 μl) was added and the viability test PMS/MTS was carriedout as described above.

(I) Activation-Induced Cell Death

Flatdish formed microtiter plates were coated with anti-human CD3 TR66(10 μg/ml) in PBS for a period of 3 hours at 37° C. The plates werewashed twice in PBS and once with RPMI 1640 medium. Jurkat cells (5×10⁵cells per ml, 100 μl) were mixed with the inhibitors and distributedover each well, then centrifuged (200×g, 3 min), and incubated for aperiod of 24 hours at 37° C. The viability of the cells (at OD 490 nm)was measured as described above. The specific cell protection (in %) wascalculated as follows: [(anti-CD3+inhibitor)−anti-CD3)]/[(control)−(anti-CD3)]×100.

For providing a mixed-leukocyte culture, splenocytes fromperforin-deficient or gld C57BL/6 mice (H-2^(b)) were cultured withgamma-irradiated (36 Gy) splenocytes from Balb/c-mice (H-2^(d)) for aperiod of 5 days. Before use, non-viable cells were removed from thesamples by gradient centrifugation on Ficoll-paque (from PharmaciaBiotech). The labelling was carried out according to the methodsdescribed herein-above (Kataoka et. al.). Briefly, target cells (A20cells) were labelled with natrium [⁵¹Cr] (from Dupont, Boston, Mass.)for a period of 1 hour, then washed three times with RPMI 1640. MLCcells were mixed with the target cells (10⁴ cells per well) in U-shapedmicrotiter plates in the presence or absence of Fas:Fc and Fas:COMP at40 μg/ml in a final volume of 200 μl and the plates were centrifuged(200×g, 3 min.). After an incubation of 4 hours, the Supernatants wereremoved and the radioactivity thereof was measured. The specific [⁵¹Cr]release (in %) was determined by the following formula: [(experimentalrelease−spontaneous release)/(maximum release−spontaneous release)]×100.

(K) FACS-Labelling

For FACS-labelling, the CD40L⁺-Jurkat Clone D1.1 (5×10⁵ cells) wasincubated with 2 μg of CD40:COMP in FACS buffer (PBS, 10% fetal calfserum, and 0.02% NaN₃).

Fas:COMP were used as negative control. Receptor:COMP was detected by 1μg of 9E10 anti-myc antibody and subsequent treatment with FITC-labelledgoat-anti-mouse antibody (1:100). Incubations were carried out for aperiod of 20 min at 4° C. in 50 μl FACS buffer.

(L) B Cell Proliferation Assay

For the assay of B cell proliferation, CD19⁺-cells from human peripheralblood lymphocytes (PBL) were purified by magnetic beads and theremaining CD19⁻ cells were irradiated (3000 rad). 10⁵ purified CD19⁺cells were mixed with 10⁵ CD19⁻ autologous irradiated PBL in 120 μl ofmedium which contained sCD40 L at a concentration of 100 ng/ml (1.8 nM),M2-antibody at 10 μg/ml plus or minus protein A at a concentration of 1μg/ml and the indicated concentrations of CD40:Fc or CD40:COMP. Then,the cells were cultivated for 72 hours in 96 well plates, pulsed with[³H] thymidine (1 μCi/well) for 6 hours and harvested. The incubation of[³H] tymidine was checked by liquid scintillation counting.

7.2 Results of the 7^(th) Embodiment

Fusion proteins which consist of extracellular domains of a receptorbeing fused to the Fc-part of IgG (receptor:Fc) are known in the art asinhibitory agents for the investigation of receptor ligand interactions.In the 7th embodiment, the size of purified Fas:Fc-fusion protein wasexamined by exclusion chromatography and it was found that a single peakwas eluted having the expected retention time. Fas:Fc containingfractions were able to protect A20-cells which were exposed to a lethaldose of soluble FasL (sFasL) against cell death although the degree ofprotection (up to 50%) was extraordinarily low, when taking intoconsideration that the estimated ratio of Fas:Fc to FasL in theexperiment was about 1000.

A weak protective activity was observed in several of the earlyfractions of the eluate, which probably contained minor nondetectableamounts of high molecular weight complexes of Fas:Fc. According to thepresent invention it was recognized that such higher aggregates of thefusion protein can act as potent inhibitors of FasL-induced cell death.Therefore, this high molecular weight fraction of aggregated Fas:Fc wasinitially raised by adding the immunoglobulin crosslinking agent proteinA. As soon as the analysis was carried out under conditions where onlyabout 10% of the injected Fas:Fc were shifted to the early elutingfractions, it was apparent that a high molecular weight Fas:Fc complexis an effective antagonist of the cytotoxicity induced by FasL. Comparedto that, it was found that the remaining Fas:Fc fractions still elutedas a dimer and only a partial protection was awarded to the cells inspite of a ten-fold higher concentration. According to the presentinvention the results of the 7th embodiment show that the formation ofhigher Fas:Fc aggregates substantially increases the specific protectiveactivity.

Therefore, according to the present invention complexes of fusionproteins were constructed on the basis of these findings, which show abetter avidity with respect to FasL and improve the inhibitorycharacteristics due to an increase in the degree of oligomerisation.Fusion proteins according to the present invention are, e.g. such fusionproteins in which the extracellular domains of receptors of the TNFfamily, e.g. Fas, TRAIL-R1, TRAIL-R2, TRAILR-3, TNF-R1 or CD40, arefused to the oligomerised domains of either the so-called cartilageoligomeric protein ((COMP); fusion protein designated as: Receptor:COMP)or the so-called cartilage matrix protein ((CMP); fusion protein:Receptor:CMP) via a 14 aminoacid-long linker. These matrix proteins andtheir domains, respectively, have the native property of formingpentameric and trimeric, respectively, coiled-coil-structures. Theabove-mentioned fusion proteins (as well as Receptor: Fc fusion proteinsas controls) were expressed in mammalian cells and purified by affinitychromatography on metalchelate columns or on protein A. In the 7thembodiment, the receptors Fas and TRAIL-R2 were also bounded to theC-terminal dimerisation domain of the protein osteoprotegrin of the TNFfamily (Receptor: δN-OPG).

The receptors which were fused to COMP or CMP oligomerized as was shownby slow migration in the polyacrylamide gel under non-reducingconditions. Fas:COMP and TRAIL-R2:CMP eluted as well-defined peakshaving apparent molecular weights of about 400 and 170 kDA,respectively, by application of the gel permeation chromatographymethod. The molecular weights correspond to pentameric and trimeric,respectively, structures of the fusion proteins. Therefore, it can beconcluded from the experiments of the 7th embodiment that theaggregation characteristics of coiled-coil oligomerisation domains ofthe above-mentioned matrix proteins is not impeded by the fusion toproteins (or protein domains) of the TNF receptor family.

The fusion protein Fas:COMP showed a lower K_(d) than Fas:Fc when thefusion proteins were allowed to compete with applied Fas:Fc for thesFasL-binding. In agreement to this result, a dissociation constant of0.77 nM was measured for the FasL-Fas:COMP interaction, which is about8- to 9-fold lower than comparative values for Fas:Fc. For the vastmajority of the cell lines tested it was shown that the inhibitoryactivity of Fas:COMP is about 10- to 20-fold higher than that for thedimeric fusion protein Fas:Fc, whereas the results for Fas:Fc aggregatescaused by the cross linking protein A (Fas:Fc/PA) showed values betweenthose for Fas:COMP and Fas:Fc. The protective activity of dimericFas:δN-OPG fusion protein was comparable to that of the dimeric Fas:Fccomplex. Trimeric Fas:CMP inhibited the sFasL-mediated lysisapproximately as effectively as Fas:Fc/PA—thus, as a result 5-fold lesseffective than Fas:COMP. The inhibitory activity of Fas:COMP was good orbetter than that of FasL-blocking monoclonal antibodies Nok-1, 4H9 and4A5. In comparison to the Fas:Fc complex, the superior inhibitoryactivity of Fas:COMP was also evident from experiments with primarymurine hepatocytes or from a model system for the activation-inducedcell death with anti-CD3-activated Jurkat cells. In all of theseexperiments a medium protection level resulted for Fas:Fc-PA.Furthermore, it was examined whether Fas:COMP is capable of inhibitingthe effect of FasL expressed on CTLs. The death of A20-cells in a 4 hourtest system is solely depended on perforin and the FasL-dependent signaltransduction pathways, since CTL which are deficient for perforin aswell as for FasL have no effect on these cells. In a correspondingexperiment, A20 cells were killed by perforin-deficient CTLs as well asby FasL-defizient CTLs, as expected. Fas:Fc and Fas:COMP specificallycaused a certain degree of protection for the cells which were exposedto perforin-deficient CTLs.

In a comparison of the affinities of sCD40L to CD40:Fc, CD40:Fc/PA orCD40:COMP according to the present invention by competitive ELISA, asubstantial increase in the affinity (30-fold) was observed for thepentamerised receptor while again a medium effect occurred forCD40:Fc/PA (8-fold). With respect to the question of whether CD40: COMPis also able to recognise membrane-bound CD40L, the Jurkat-derivatisedcell line D1.1, which expresses the surface protein CD40Lconstitutively, was used for the oligomerisation of a FACS labellinganalysis with the aid of the CD40 fusion proteins as samples. Asignificant labelling was observed for CD40:COMP according to thepresent invention, which indicates that CD40-COMP is in fact capable ofbinding native unprocessed CD40L. In order to examine the specificactivity of CD40 fusion protein in a biological system, it was attemptedto inhibit the CD40L-dependent proliferation of human B cells which wereco-stimulated with anti-B-cell receptor. Neither CD40:Fc nor CD40:Fc/PAwere able to impede the proliferation significantly even in the casewhere they were administrated in high doses. On the contrary, CD40:COMPaccording to the present invention was capable of blocking theproliferation already at relative low doses.

Summing up, it can be concluded from the observations of the presentembodiment that the inhibition of FasL-induced apoptosis by competitiveinhibitors increased depending on the degree of oligomerisation and thatthe relative activities of the various inhibitors (expressed as theproportion of their respective IC₅₀ values) remained relatively constantfor the various cell lines. The increased inhibitory activity ofFas:COMP according to the present invention in comparison to Fas:Fc isprobably the result of its higher avidity. Similar results were alsodetermined for the activity of the CD40 fusion protein according to thepresent invention. CD40:COMP according to the present invention isdistinctly superior to CD40:Fc in vitro with respect to the inhibitionof CD40L-induced proliferation of primary B cells. Accordingly, theresults of embodiment 7 show that dissociation constants in the lownanomolar region can be obtained for such receptors as, e.g. Fas andCD40, which are natively characterised by a medium affinity for theirligands, if they, as intended by the present invention, are a componentof fusion proteins which are characterised by a higher degree ofoligomerisation.

1. A bimer or oligomer of dimers, trimers, quadromers or pentamers ofrecombinant fusion proteins characterized by the recombinant fusionproteins having at least one component A and at least one component B,wherein component A comprises a protein or a protein segment withbiological function, in particular with ligand function for antibodies,for soluble or membrane-bound signal molecules or for receptors or anantibody or segment of an antibody and component B comprises a proteinor a protein segment selected from the group consisting of the family ofC1q proteins and the collecting, which bimerizes or oligomerizes thedimer, trimer, quadromer or pentamer of the recombinant fusion proteinwithout the effect of third molecules.
 2. The bimer or oligomer ofdimers, trimers, quadromers or pentamers of recombinant fusion proteinsaccording to claim 1 characterized by the component A of the recombinantfusion proteins in the dimer, trimer, quadromer or pentamer beingidentical or different.
 3. The bimer or oligomer of dimers, trimers,quadromers or pentamers of recombinant fusion proteins according toclaim 1 characterized by the component A of the recombinant fusionproteins being a peptide hormone, a growth factor, a cytokine, aninterleukin, a segment thereof or a functional derivative of theabove-mentioned sequences.
 4. The bimer or oligomer of dimers, trimers,quadromers or pentamers of recombinant fusion proteins according toclaim 1 characterized by the component A of the recombinant fusionproteins being a cytokine from the family of TNF cytokines, a segment ofsuch a TNF cytokine or a functional derivative of the above-mentionedsequences.
 5. The bimer or oligomer of dimers, trimers, quadromers orpentamers of recombinant fusion proteins according to claim 4characterized by the component A of the recombinant fusion proteinsbeing a TNF cytokine or a segment of a TNF cytokine selected from thegroup consisting of CD40L, FasL, TRAIL, TNF-a, CD30L, OX40L, RANKL,TWEAK, Lta, Ltab2, LIGHT, CD27L, 41-BB, GITRL, APRIL, EDA, VEGI andBAFF, or a functional derivative of the above-mentioned sequences. 6.The bimer or oligomer of dimers, trimers, quadromers or pentamers ofrecombinant fusion proteins according to claim 1 characterized by thecomponent A of the recombinant fusion proteins being an antigen or asegment of an antigen of viral, bacterial or protozoological pathogens.7. The bimer or oligomer of dimers, trimers, quadromers or pentamers ofrecombinant fusion proteins according to claim 6 characterized by thecomponent A of the recombinant fusion proteins being a surface antigenor a segment of a surface antigen of a viral, bacterial orprotozoological pathogen.
 8. The bimer or oligomer of dimers, trimers,quadromers or pentamers of recombinant fusion proteins according toclaim 1 characterized by the component A of the recombinant fusionproteins being an amino acid sequence coupled to a receptor agonist orto a receptor antagonist.
 9. The bimer or oligomer of dimers, trimers,quadromers or pentamers of recombinant fusion proteins according toclaim 1 characterized by the component B of the recombinant fusionproteins containing a multimerizing and oligomerizing segment or afunctional derivative of such a segment of a protein, selected from thegroup consisting of C1q, MBP, SP-A, SP-D, BC, CL43 and ACRP30, orcontaining the collagen domain of the EDA protein or a functionalderivative thereof.
 10. The bimer or oligomer of dimers, trimers,quadromers or pentamers of recombinant fusion proteins according toclaim 9 characterized by the component B containing an amino acidsequence, as shown in FIG. 6A for the sequence of amino acids 18 to 111of mACRP30 or a functional derivative thereof, or containing an aminoacid sequence, as shown in FIG. 6B for the sequence of amino acids 18 to108 of hACRP30 or a functional derivative thereof, whereby FIGS. 6A and6B, respectively is part of the claim.
 11. The bimer or oligomer ofdimers, trimers, quadromers or pentamers of recombinant fusion proteinsaccording to claim 1 characterized by the component B containing asequence of 8 to 20 amino acids which bimerizes or oligomerizes dimersor multimers of recombinant fusion proteins.
 12. The bimer or oligomerof dimers, trimers, quadromers or pentamers of recombinant fusionproteins according to claim 11 characterized by the component B formingone or more disulfide bridges.
 13. The bimer or oligomer of dimers,trimers, quadromers or pentamers of recombinant fusion proteinsaccording to claim 12 characterized by the component B containing theamino acid sequence LEGSTSNGRQCAGIRL or a functional derivative of thissequence.
 14. The bimer or oligomer of dimers, trimers, quadromers orpentamers of recombinant fusion proteins according to claim 11characterized by the component B containing the sequenceLEGSTSNGRQSAGIRL or a functional derivative of this sequence.
 15. Thebimer or oligomer of dimers, trimers, quadromers or pentamers ofrecombinant fusion proteins according to claim 1 characterized by themultimerized and oligomerized fusion proteins having antigenic segmentsfor detecting and crosslinking by antibodies.
 16. The bimer or oligomerof dimers, trimers, quadromers or pentamers of recombinant fusionproteins according to claim 1 characterized by the recombinant fusionproteins having a linker sequence between component A and component B.17. The bimer or oligomer of dimers, trimers, quadromers or pentamers ofrecombinant fusion proteins according to claim 1 characterized by therecombinant fusion proteins having two or more different components B.18. A medicament comprising a pharmaceutical composition of a bimer oroligomer according to claim
 1. 19. Method of treatment of a disorder ordisease comprising administration of a bimer or oligomer according toclaim wherein the disorder or disease is a hyperinflammatory disorders,autoimmune diseases, diseases based on hyperapoptotic or hypoapoptoticdisorders, infectious diseases, viral infectious diseases, tumors, inparticular tumors of the lymphatic system, and/or endocrinologicaldisorders.
 20. A vaccine comprising a bimer or oligomer according toclaim 1 wherein the vaccine is for active or passive immunizationagainst infectious diseases.
 21. A vaccine according to claim 20 forvaccination against German measles, measles, poliomyelitis, rabies,tetanus, diphtheria, BCG, malaria, yellow fever, HIV or influenza.
 22. Avaccine according to claim 20 which is formulated for parenteral or oraladministration.
 23. A method for in-vitro diagnosis comprising applyinga bimer or oligomer according to claim 1 to an in-vitro protein-basedderivative.
 24. A fusion protein characterized by the recombinant fusionprotein containing a component A and a component B, whereby component Acontains a protein or a protein segment with biological function, inparticular ligand function for antibodies or receptors or an antibody orsegment of an antibody or for soluble or membrane-bound signalmolecules, and component B contains a multimerizing and oligomerizingsegment or a functional derivative of such a segment of a protein,selected from the group consisting of the family of the C1q proteins andthe collecting.
 25. The fusion protein according to claim 24characterized by the component B of the fusion protein containing amultimerizing and oligomerizing segment of a protein or a functionalderivative of the same, selected from the group consisting of C1q, MBP,SP-A, SP-D, BC, CL43 and ACRP30, or containing the collagen domain ofthe EDA protein or a functional derivative thereof.
 26. A DNA sequencecharacterized by the DNA sequence encoding the fusion protein accordingto claim
 24. 27. An expression vector characterized by the expressionvector containing the DNA sequence according to claim
 26. 28. A hostcell characterized by the host cell being transfected with an expressionvector according to claim
 27. 29. A soluble multimeric polypeptide of atleast two trimer units, wherein each trimer unit comprises a fusionprotein trimer strand consisting of: a first polypeptide comprising thefirst about 100 to 250 N-terminus residues of a collectin familyscaffold protein, wherein the first polypeptide comprises a hub and abody region of the collectin family scaffold protein; and a secondpolypeptide comprising the last about 100 to 250 C-terminus residues ofa tumor necrosis factor superfamily (TNFSF) ligand, wherein the secondpolypeptide comprises an extracellular domain (ECD) of the TNFSF ligand,wherein the carboxy-terminal residue of the first polypeptide isoperably linked to the amino-terminal residue of the second polypeptidevia: i) deletion of a carbohydrate recognition domain (CRD) of thecollectin family scaffold protein and ii) replacement of the CDR withthe ECD of the TNFSF ligand, whereby a single trimer strandspontaneously trimerizes with two additional trimer strands to form atrimer unit and the trimer unit binds at the hub to form the multimericpolypeptide.
 30. The multimeric polypeptide of claim 29, wherein theTNFSF ligand is selected from lymphotoxin-A (LTA), lymphotoxin-B (LTB),tumor necrosis factor (TNF), or any of TNFSF4-15 and TNFSF18.
 31. Themultimeric polypeptide of claim 29, wherein the collectin familyscaffold protein is selected from complement factor 1 (C1q), mannosebinding protein, mannose-binding lectin type 1 (MBL1), mannose-bindinglectin type 2 (MBL2), pulmonary surfactant protein A (SPA), pulmonarysurfactant protein D (SPD), conglutinin, collectin 43, C-type lectin L1(CL-L1), adipocyte complement related protein of 30 kDa (ACRP30), orhibernation specific protein 27 (Hib27).
 32. The multimeric polypeptideof claim 29, wherein the trimer unit comprises homomeric trimer strands.33. The multimeric polypeptide of claim 29, wherein the trimer unitcomprises heteromeric trimer strands.
 34. The multimeric polypeptide ofclaim 29, wherein the collectin family scaffold protein is surfactantprotein D.
 35. The multimeric polypeptide of claim 29, wherein the TNFSFligand is CD40L.
 36. The multimeric polypeptide of claim 29, wherein thetrimer strand is SPD-CD40L.
 37. The multimeric polypeptide of claim 29,wherein the TNFSF ligand is receptor activator of NF-kappaB ligand(RANKL).
 38. The multimeric polypeptide of claim 29, wherein the trimerstrand is SPD-RANKL.
 39. The multimeric polypeptide of claim 29, whereinthe TNFSF ligand is CD27L/CD70.
 40. The multimeric polypeptide of claim29, wherein the trimer strand is SPD-CD27L/CD70.
 41. The multimericpolypeptide of claim 29, wherein amino acid residues comprising thetrimerized strands which are susceptible to proteolytic degradation areremoved from the multimeric polypeptide.
 42. The multimeric polypeptideof claim 29, wherein the multimer is a dimer of trimer units.
 43. Asoluble multimeric polypeptide of at least two trimer units, whereineach trimer unit comprises a fusion protein trimer strand comprising afirst polypeptide comprising a multimerizing and oligomerizing segmentof an extracellular matrix protein having a carbohydrate recognitiondomain (CRD) and wherein the protein is selected from the groupconsisting of the family of C1q proteins and collectin family proteins;and a second polypeptide of a membrane-bound signal protein comprisingan extracellular domain (ECD), wherein the carboxy-terminal residue ofthe first polypeptide is operably linked to the amino-terminal residueof the second polypeptide via: i) deletion of a carbohydrate recognitiondomain (CRD) of the first polypeptide and ii) replacement of the CRDwith the ECD of the second polypeptide, whereby a single trimer strandspontaneously trimerizes with two additional trimer strands to form atrimer unit and the trimer unit binds to form the multimericpolypeptide.
 44. The multimeric polypeptide of claim 43 wherein theextracellular matrix protein is cartilage oligomeric matrix protein(COMP) or cartilage matrix protein (CMP), C1q, collagen α1(X), collagenα2(VII), ACRP30, the internal ear structure protein, cerebelin,multimerin, Lung surfactant protein (SP-A), mannose binding protein(MBP), lung surfactant protein (SP-D), bovine serum conglutinin (BC), orbovine collectin-43 (CL43).
 45. The multimeric polypeptide of claim 43wherein the membrane-bound signal protein comprising an extracellulardomain (ECD) is a TNF cytokine.
 46. The multimeric polypeptide of claim45 wherein the TNF cytokine is CD40L, FasL, TRAIL, TNF, CD30L, OX40L,RANKL, TWEAK, Lta, Ltab2, LIGHT, CD27L, 41-BB, GITRL, APRIL, EDA, BEGIor BAFF.
 47. The multimeric polypeptide of claim 45 further comprising apeptide linker sequence between the first and second polypeptide.